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(i)

TO MY PARENTS

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/ (ii)

BOTULINUM NEUROTOXINS AND THEIR NEURONAL ACCEPTORS r

» Richard Stephen Williams

Department of Biochemistry

Imperial College of Science and Technology

* University of London

A dissertation submitted for the t degree of Doctor of Philosophy of

the University of London

and the Diploma of Imperial College 4

April, 1984 (iii)

“Poisons can be employed as means for the destruction of life or as

agents for the treatment of the sick, but in addition to these two well

recognised uses there is a third of particular interest to the

physiologist. For him the poison becomes an instrument which

dissociates and analyses the most delicate phenomena of living

structures and by attending carefully to their mechanism in causing

death he can learn indirectly much about the physiological processes of

life." *

(Claude Bernard)

* ABBREVIATIONS

ACh Acetylcho1ine

AChE Acetylcholi ne esterase

AChR Acetylcholine receptor

AMP, ADP and ATP Adenosine mono-, di- and tri-phosphate, respectively

BAEE Na-benzoyl-L-arginine ethyl ester bis NN'-Methylenebi sacryl ami de

B1WSV Black Widow Spider Venom

BoNT Botulinum neurotoxin

BrWSV Brown Widow Spider Venom

BuTx Bungarotoxin

CAM Carboxyami do-methylated

CAT Choline acetyl transferase

Cl. Clostri di urn

CNS Central nervous system

CRM Cross-reactive forms

ConA Concanavalin A

OEAE Diami noethane tetraacetic acid

DTT Dithiothreitol

DTx Dendrotoxi n

EDTA Diaminoethanetetraacetic acid, disodium salt

EF-2 Elongation factor-2

EGTA Ethyleneglycol-bi s-(3-ami noethyl ether) N,N'-tetra-

acetic acid

ELISA Enzyme-1 inked-immunosorbent assay epp End-plate potential

GDP and GTP Guanosine di- and tri-phosphate, respectively

GERL Go!gi-endoplasmic reticulum-lysosome

GTPase Guanosine triphosphatase m

GuHCl Guanidinium hydrochloride

HPLC High pressure liquid chromatography 125 125I-Bo MT I-labelled botulinum neurotoxin

Dissociation constant k d

LD50 Lethal dose 50 mepp Miniature end-plate potential

MeV Mega electron volt

Relative molecular weight M r NAD (P) Nicotinamide dinucleotide (phosphate), oxidised form

#* NADH, NADPH Reduced forms of NAD, NADP

NAG N-acetylglucosamine

NANA N-acetylneuraminic acid

% NMJ Neuromuscular junction PAGE Polyacrylamide gel electrophoresis

Pi Isoelectric point

p l a 2 Phospholipase A2 I PMSF Phenyl methyl sulphonyl fluoride

QAE Di ethyl -(2-hydroxypropyl) ami noethyl

Q10 Temperature coefficient RCAj Ricinus communis agglutinin (type I)

SBA Soyabean agglutinin

SDS Sodium dodecyl sulphate

% TEMED N,N,N',N'-Tetramethylethylene diamine TLE Trypsin-like enzyme

WGA Wheat germ agglutinin (vi)

CONTENTS

Page

Abbreviations ...... (iv)

Table of Contents ...... (vi)

• List of Tables ...... (xi)

List of Figures ...... (xii)

Acknowl edgements ...... (xvi)

Abstract ...... 1 * Chapter 1 General Introduction ...... 3

1.1 Neurotransmi ssi on ...... 4

1.1.1 Electrical and chemical synapses...... 4

* 1.1.2 Modes of neurotransmitter release...... 8

1.1.2.1 Quantal release of transmitter...... 8

1.1.2.2 Non-quantal release of transmitter...... 13

£ 1.2 Botulism...... 14

1.3 Toxins from Cl. botulinum ...... 16

1.3.1 Botulinum toxin-haemagglutinin complexes...... 16

1.3.2 Structure and activity of the neurotoxins...... 17 * 1.3.3 Bacteriophages and toxigenicity of Cl. botulinum...... 20

1.4 The action of botulinum toxins ...... 21

1.4.1 Interaction with neuronal tissues...... 21

m 1.4.2 Pharmacological actions of botulinum toxins...... 23

1.5 Other bacterial and plant toxins ...... 33

1.5.1 Cholera toxin...... 33

* 1.5.2 Diphtheria toxin...... 34

1.5.3 Tetanus toxin...... 36

1.5.4 Abrin and ricin...... 38

1.6 Presynaptically active snake venom toxins ...... 40

1.6.1 Snake toxins exhibiting phospholipase A2 activity...... 40

1.6.2 Venom toxins without PLAg activity...... 43 1.7 The present study ...... 44 (Vii)

Page

Chapter 2 Purification of neurotoxin from Clostridium

botulinum types A and B ...... 46

2.1 Introduction ...... 47

* 2.2 Methods ...... 49

2.2.1 General safety...... 49

2.2.2 Purification of neurotoxin from Cl. botulinum type B

by affinity and ion-exchange chromatography...... 50 * 2.2.3 Isolation of type A neurotoxin by affinity and ion-

exchange chromatography...... 51

2.2.4 Native and SOS gradient-pore gel electrophoresis...... 52

♦ 2.2.5 Two-dimensional gel electrophoresis...... 53

2.2.6 Ouchterlony double immunodiffusion...... 54

2.2.7 QAE-Sephadex chromatography of type A BoNT...... 54

2.2.8 Other determinations...... 55 * 2.3 Results ...... 55

2.3.1 Purification of type B BoNT...... 55

2.3.1.1 Affinity chromatography of crude

toxi n-haemaggl uti ni n compl exes...... 55

2.3.1.2 DEAE-Sephacel chromatography of

affinity purified material...... 60

* 2.3.2 Purification of type A BoNT...... 64

2.3.3 Separation of subunits from BoNT (A) byQAE-Sephadex

chromatography...... 72

^ 2.4 Discussion ...... 79

Chapter 3 Structural characteristics of BoNT types A and B...85

3.1 Introduction ...... 86 (viii)

Page

3.2 Methods ...... 91

3.2.1 Preparation of subunits from BoNT (Aand B) by SDS-PAGE...... 91

3.2.2 Amino-acid analysis...... 92

% 3.2.3 Peptide mapping by limited proteolysis

in SDS-acrylamide gels...... 93

3.2.4 Silver staining of SDS-acryl amide gels...... 94

3.2.5 Preparation of subunits from carboxyamidomethylated (CAM)-

BoNT (A and B) by high pressure liquid chromatography (HPLC)...95

3.2.6 Peptide mapping of CAM-subunits by reverse-phase HPLC...... 95

3.2.7 Protein determination in the presence of SDS...... 96

* 3.3 Results ...... 97

3.3.1 Isolation of subunits from types A and B BoNT by

preparative SDS-PAGE...... 97

k 3.3.2 Amino-acid compositions of subunits

from native BoNT (A and B)...... 101

3.3.3 Electrophoretic peptide mapping of constituent

subunits of types A and B neurotoxins...... 107 * 3.3.4 Rapid preparation of polypeptides from CAM-BoNT by HPLC...... 108

3.3.5 Comparison of reverse-phase HPLC peptide maps

obtained from CAM-subunit digests...... 113

* 3.4 Discussion ...... 116

Chapter 4 Radioiodination of types A and B BoNT:

their specific interactions with

cerebrocortical synaptosomes...... 123

4.1 Introduction ...... 124

4.2 Methods ...... 126

4.2.1 Radiolabelling of botulinum neurotoxins...... 126 1 oc 4.2.2 Characterisation of I-BoNT (A and B)...... 127 (ix)

Page

4.2.3 Separation of native toxin from its labelled species...... 127

4.2.4 Preparation of rat cerebrocortical synaptosomes...... 128 125 4.2.5 Measurement of I-BoNT (A and B) binding to synaptosomes. ..129

* 4.2.6 Effect of temperature and pH on synaptosomal

binding of type A 125I-BoNT...... 130

4.2.7 Electron-microscope autoradiography ofsynaptosomes

labelled with 125I-BoNT (A)...... 131 * 4.3 Results ...... 134

4.3.1 Properties of type A *2^I-BoNT...... 134

4.3.2 Separation of native toxin from itslabelled species...... 138

* 4.3.3 Saturable binding of *2!*i-BoNT (A) to rat

cerebrocortical synaptosomes...... 145

4.3.4 Effect of temperature and pH on synaptosomal

^ binding of type A *^I-BoNT...... 153 125 4.3.5 Location of acceptor(s) for type A I-BoNT

on synaptic membranes...... 153

4.3.6 Characterisation of acceptor sites for type B * 125 I-BoNT on synaptosomal membranes...... 157

4.4 Discussion ...... 165

Chapter 5 Nature and selectivity of neuronal receptors

for BoNT...... 172

5.1 Introduction ...... 173

5.2 Methods ...... 176

5.2.1 Investigations into the nature ofacceptors for 1 25 for types A and B ^ I - B o N T ...... 176

5.2.2 High-energy radiation of synaptosomalmembranes

and standard enzyme markers...... 179 m

Page

5.2.3 Studies on the selectivity of synaptosomal 1 ?5 acceptors for I-BoMT (A and B)...... 181 125 5.2.4 Preparation of I-labelled subunits from type A BoMT...... 132

5.3 Results ...... 184

5.3.1 Nature of saturable acceptors for types A and B 125 I-BoNT on synaptosomal membranes...... 184 1 5.3.2 Target size analysis of acceptors for I-BoNT (A and B). ...196

5.3.3 Selectivity of synaptosomal acceptorsfor

types A and B 125I-BoNT...... 196

5.3.4 Preliminary studies on the preparation of 125 I-labelled subunits...... 205

5.4 Discussion ...... 208

* Chapter 6 General Discussion...... 217

6.1 Botulinum neurotoxins - an homologous group ...... 218

6.2 Sequential stages in the action of BoNT ...... 223

6.2.1 Toxin binding...... 224 * 6.2.2 Internalisation...... 230

6.2.3 Toxin-induced inhibition of neurotransmitter release...... 233

6.3 Similarities with tetanus toxin ...... 235 » 6.4 Future perspectives ...... 239

Appendix ...... 243

References ...... 244 (Xi)

List of Tables

Page

Table 2.1 Purification of Cl. botulinum type B neurotoxin...... 56

* Table 2.2 Purification of Cl. botulinum type A neurotoxin...... 65

Table 2.3 Separation and reconstitution of subunits from

type A BoNT...... 76

Table 2.4 Summary of the structural properties of neurotoxins

from Cl. botulinum types A and B ...... 83

Table 3.1 Amino-acid compositions of the subunits from type

A BoNT...... 99

Table 3.2 Amino-acid compositions of the subunits from type

B BoNT...... 100 125 Table 4.1 Conditions for I-iodination of type A BoNT

using the chloramine-T method...... 133 1 25 Table 4.2 Binding of I-BoNT (A) to synaptosomal

membraneous fractions...... 154

Table 5.1 Nature of the high-affinity synaptosomal acceptor

for BoNT (A)...... 185

Table 5.2 Selectivity of the high-affinity acceptor(s) for 125 I-BoNT (A and B) on synaptosomal membranes...... 198 *

* List of Figures

Page

Fig. 1.1 Diagramatic representation of the frog

neuromuscular junction...... 6

Fig. 1.2 Schematic representation of possible mechanisms

of ACh release at the synapse...... 12

Fig. 1.3 Generalised structure of botulinum neurotoxins and

possible pathways of nicking and activation...... 18

Fig. 1.4 Schematic diagram of a nerve terminal illustrating

the two types of vesicular transmitter release (I and

II) as proposed by Thesleff...... 29

Fig. 2.1 Affinity and ion-exchange chromatography of type B

toxi n-haemaggl uti nin compl exes...... 57

Fig. 2.2 SDS-PAGE of BoNT (B) at different stages of

purification...... 59

Fig. 2.3 Ouchterlony double immunodiffusion gels of proteins

purified from type B toxin complexes...... 61

Fig. 2.4 Native gel electrophoresis of type B BoNT at

different stages of purification...... 62

Fig. 2.5 Two-dimensional gel electrophoresis of type B BoNT...... 63

Fig. 2.6 Affinity and DEAE-Sephacel chromatography of toxin

complexes from Cl. botulinum type A ...... 66

Fig. 2.7 Analysis of BoNT (A) at different stages of

purification by PAGE...... 68

Fig. 2.8 Ouchterlony double immunodiffusion of proteins

obtained during purification of BoNT (A) from

toxi n-haemaggl uti nin complexes...... 71

Fig. 2.9 QAE-Sephadex chromatography of type A BoNT under

reducing conditions in the presence of urea...... 73 (xiii)

Page

Fig. 2.10 SDS-PAGE of subunits from BoNT (A) separated by

QAE-Sephadex chromatography...... 75

Fig. 2.11 Ouchterlony double immunodiffusion of type A neurotoxin

► subunits prepared by QAE-Sephadex chromatography...... 78

Fig. 3.1 Cross-sectional view of the apparatus used for

electroelution of proteins from acrylamide gels...... 89

Fig. 3.2 Purity of subunits obtained from native BoNT, ¥ type A and B, by preparative SDS-PAGE...... 98

Fig. 3.3 Comparison between electrophoretic peptide maps of

subunits obtained from types A and B BoNT...... 103

Fig. 3.4 Similarities in the peptide maps of subunits

from CAM-BoNT (A and B)...... 105

Fig. 3.5 Rapid preparation of subunits from CAM-BoNT types

A and B by HPLC under denaturing conditions...... 109 ♦ Fig. 3.6 Purity of the subunits obtained by HPLC...... Ill

Fig. 3.7 Reverse-phase HPLC peptide maps of polypeptides

prepared from CAM-BoNT type B ...... 114 * Fig. 3.8 Comparison betweeen reverse-phase HPLC peptide maps of

alkylated H-subunits from types A and B neurotoxins. ...115

Fig. 4.1 Sephadex G-25 chromatography of type A 125I-BoMT...... 132 41 125 Fig. 4.2 Autoradiographic analysis of I-BoNT (A)

following SDS-PAGE...... 135 125 Fig. 4.3 Immunoreactivity of native and I-BoNT (A)...... 136 1 25 * Fig. 4.4 Isoelectricfocussing of type A I-BoNT...... 137 1 pc Fig. 4.5 Chromatofocussing of type A I-BoNT...... 139 125 Fig. 4.6 Anion-exchange chromatography of I-BoNT (A)...... 141 1 25 Fig. 4.7 Equilibrium binding of type A I-BoNT to

purified rat cerebrocortical synaptosomes. 143 (XiT)

Page 1?5 Fig. 4.8 Association of type A I-BoNT with acceptor

sites on synaptosomes...... 146

Fig. 4.9 Dissociation of toxin from synaptosomes prelabelled

* with 125I-Bo NT (A)...... 149

Fig. 4.10 Effect of temperature and pH on synaptosomal

binding of type A 12J*I-BoNT...... 151

Fig. 4.11 Electron-microscope autoradiographic localisation of

* IOC the I-BoNT (A) binding component on synaptosomes. ...155

Fig. 4.12 SDS-PAGE of type B 125I-BoNT...... 158

Fig. 4.13 Equilibrium binding of *2^I-BoNT (B) to

* cerebrocortical synaptosomes...... 159 125 Fig. 4.14 Association of type B I-BoNT with acceptors on

synaptosomal membranes...... 161

^ Fig. 4.15 Dissociation of toxin from synaptosomes prelabelled

with 125I-Bo NT (B)...... 163

Fig. 5.1 Heat and trypsin sensitivity of neuronal acceptor(s)

for 125I-Bo NT (B)...... 186 1 Fig. 5.2 Effect of neuraminidase on synaptosomal binding of

types A and B 125I-BoNT...... 189 125 Fig. 5.3 Interaction of type B I-BoHT with gangliosides...... 190 M 125 Fig. 5.4 Effect of lectins on the binding of I-BoNT

(A and B)...... 191 125 Fig. 5.5 Target size analysis of acceptors for I-BoNT

+ (A and B)...... 194

Fig. 5.6 Effect of type B BoNT and subunits from type A BoMT on

125I-Bo NT (A) binding...... 197

Fig. 5.7 Effect of nicked BoNT (B),BoNT (A), 3-bungaro-

toxin and dendrotoxin on binding of type 8 125 I-BoNT to synaptosomes...... 200 (IV )

Page

Fig. 5.8 Effect of tetanus toxin, 3-bungarotoxin, dendrotoxin

and botulism antagonists on the binding of

125I-Bo NT (A)...... 201

* Fig. 5.9 Ability of a-latrotoxin, crotoxin, taipoxin and

bee venom phospholipase Ag to interfere with

1 i-BoNT (A) binding...... 203

Fig. 5.10 Two-dimensional IEF-SDS PAGE of 125I-BoNT (A)...... 206

^ IOC Fig. 5.11 Specific binding to synaptosomes of I-labelled

subunit (M„ 91000) from BoNT (A)...... 207 r

H (XYi)

Acknowledgements

I wish to thank my supervisors, Dr. J.O. Dolly and

Prof. J. Melling for providing me with the opportunity to pursue this

very interesting line of research and also for their dedicated

supervision and encouragement over the past three years. In addition,

my thanks to: the S.E.R.C. for 3 years of financial support;

Jenny Black for her dear friendship throughout my time at Imperial

College, enjoying the good times and roughing the bad ones together;

also for her assistance in the electron microscopy; Dr. Peter Hambleton,

Dr. Clifford Shone, Brian Capel, Nigel Bailey, Roger Rhind-Tutt and

Harry Coombes of P.H.L.S., Porton Down for their indispensible

assistance, understanding and friendship during my visits; especially

to Clifford Shone for performing some of the routine toxin

purifications; Debbie Conti, also at P.H.L.S., for providing transport

for me, often at very short notice; Dr. Mike Waterfield of Imperial

Cancer Research Fund for providing laboratory facilities for some of the

peptide mapping studies and to his staff (Geoff Scrace, Rob Philp,

Nick Totty) for their expert technical help, useful discussions and

making of my time there an enjoyable one; Dr. R. Shippoloni for

assistance in performing experiments using HPLC at Imperial College;

David Green for performing injections of botulinum toxins at Imperial

College; Glen Millhouse for. expert photography; Dave Featherbe for

running the amino-acid analyser; Tony Ashton and others too numerous to mention on the 4th and 5th floors for their companionship; Liz Ashton

for the expert typing of this thesis; last, but never least, to Sally,

for being there. ABSTRACT

The presynaptically-active neurotoxins (BoNT) [M %150000] from

Cl. botulinum types A and B were purified from their crude complexes with haemagglutinin, by affinity and ion-exchange chromatography to high o levels of specific neurotoxicity (1-4 x 10 mouse LDgQ/mg protein).

Sodium-dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions showed that the nicked toxin contain two non identical subunits (PI -ulOOOOO and 50000). These subunits were r prepared from type A BoNT by ion-exchange chromatography on QAE-

Sephadex-A50 in the presence of reducing agent and urea; they showed minimal toxicity ( 1% of the native toxin but retained their immunoreactivity and could be used to reconstitute biologically active toxin.

A comparative study was made of the structures of the different subunits from types A and B BoNT. Polypeptides obtained by preparative

SDS-PAGE showed no significant differences in the relative amounts of amino-acids present; moreover, a statistical analysis indicated possible sequence homologies between the subunits of the same type of

BoNT and between similar-sized polypeptides of different neurotoxin types. Peptide mapping studies on the subunits from types A and B BoNT, by limited proteolysis in SDS-acrylamide gels or reverse-phase high pressure liquid chromatography following extensive digestion with protease, also suggest some degree of homology in the structures of, at least, types A and B BoNT.

BoNT (A and B) were radioiodinated ( I-iodine) to high specific activity (800-2000 Ci/mmol), using a modification of the chloramine-T procedure, with appreciable retention of their biological activities (60-80%). The labelled toxins were found to bind saturably to membranes of rat cerebrocortical synaptosomes; analysis of equilibrium binding data showed heterogeneous populations of acceptors

for both types A and B 125I-labelled proteins (KQ *03.5 and 20nM,

B__v %50 fmol/mg and 3pmol/mg protein, respectively). Heterogeneity of . iTlaX sites was also evident from kinetic data. The binding of type A 125 I-BoNT to synaptosomes was shown to be mediated by its larger

subunit and was totally prevented by heating the membranes (95°C) or by

treatment with trypsin. In contrast, acceptors for type B *25I-BoNT

were partially sensitive to heat and trypsinisation; at least in the

case of the heat resistent sites, these appear to be of high-affinity. 1 25 Types A and B I-BoNT were found to interact only weakly with

gangliostdes although treatment of synaptosomal membranes with

neuraminidases and lectins implicate sialic acid residues in the toxins'

acceptor!s). Target size analysis of membraneous acceptors suggest the

involvement of low molecular weight components in toxin binding. 125 I-BoNT (A and B) appear to bind selectively to distinct

sites; however, type A BoNT, at high concentrations, may partially 1 pc inhibit the binding of type B I-BoNT. Acceptors for these labelled toxins do not exhibit any interaction with some other presynaptically- active neurotoxins (e.g. 8-bungarotoxin, dendrotoxin or o-latrotoxin) or botulism antagonists (e.g. 4-ami nopyridine, chloroquine). Tetanus toxin inhibited *2^I-BoNT (A) binding, albeit with low affinity. Crotoxin and taipoxin gave a significant reduction in toxin binding but this resulted from their phospholipase activities. Therefore, it appears that highly specific synaptosomal acceptors exist for types A and B BoNT which are of low molecular weight and are directly or indirectly associated with a proteinaceous component(s). The functional significance of such acceptors) is discussed in relation to the toxins' effect on neurotransmitter release. - 3 -

♦

CHAPTER 1

GENERAL INTRODUCTION

4 1.1 NEUROTRANSMISSION

1.1.1 Electrical and Chemical Synapses. The nervous system is a highly complicated arrangement of nerve cells that exist to relay information

(via nerve impulses) between one part of the body and another. The nerve cell is composed of three main sections, the cell body, the axon and the nerve terminal. An impulse arises in or near the cell body and is conducted via the axon to the nerve terminal as an action potential, by a series of membrane depolarisation-repolarisation events due to changes in Na+ and K+ ion permeabilities in the membrane (Hodgkin and Huxley, 1952a,b). The nerve terminal is a highly specialised structure that enables an impulse to be transferred from the nerve to the organ it innervates (e.g. another nerve, muscle or gland), via a synapse. The synapse is a junction between the terminal boutons at the ends of the axonal branches of one cell and the membrane of the effector organ; the membrane of the transmitting cell is the presynaptic membrane and that of the receiving cell is the post-synaptic membrane.

In a few instances, the nerve terminal is directly coupled to the organ it innervates, with no gap occuring between the pre- and post-synaptic membranes (Robertson, 1955, 1961); in such cases transmission is by direct electrical stimulation ll-urshpan and Potter, 1959; Phillis,

1974). An example of electrical transmission is to be found in the giant nerve terminals of the squid, where it is characterised by low electrical resistance, negligible synaptic delay and responses of large amplitudes (Phillis, 1974). In the majority of cases, however, nervous transmission is a chemical process since the nerve terminal is not electrically coupled to the organ it innervates, due to the presence of a synaptic gap or cleft (about 20nm across) which separates pre-synaptic

(nerve terminal) from post-synaptic structures. The chemical links between pre- and post-synaptic regions are know/i as neurotransmitters.

When an action potential reaches the synapse it depolarises the pre-

synaptic membrane and causes a large influx of Ca ions (Katz and

Miledi, 1965a; Baker et al_., 1971; Baker, 1976); this is thought to

facilitate transmitter release from the pre-synaptic membrane at

specific sites ("active zones") into the synaptic cleft (del Castillo

and Katz, 1954; Whittaker et al_., 1963; de Roberti s et al_., 1963;

Martin, 1966; Wilson et ^1_., 19/3). ihe depolarised presynaptic membrane is able to recover to its resting state (repolarisation) by an

increase in the permeability of the membrane to K+ions. This depolarisation-repolarisation, cycle may be completed in less than 1 msec. In the resting state, small fluctuations in the postsynaptic potential, miniature endplate potentials (mepps), may be observed; these mepps reflect the release of small amounts of transmitter from the pre synaptic membrane. The transmitter substance, once in the cleft, is able to diffuse across to the postsynaptic membrane where it interacts with specific receptors. If this interaction is sufficient to cause a significant change in the postsynaptic membrane potential then an endplate potential (epp) arises to elicit its postsynaptic effect (e.g. nerve impulse conduction, muscle contraction, secretion) (Kuffler,

1948). In the case of motor nerve innervation of skeletal muscles, the transmitter is acetylcholine (ACh) and hence the term cholinergic nervous transmission has evolved. ACh either interacts with its post— synaptic receptor, the nicotinic acetylcholine receptor (AChR), and/or is inactivated by acetylcholinesterase (AChE); this enzyme cleaves ACh to choline and acetate, thus removing ACh from the synaptic cleft and leaving the AChR free to respond to subsequent stimulation (Iversen,

1971; Bennet et £l_., 1972; Kuhar, 1973). The latter product diffuses away but choline is actively taken up by the presynaptic membrane

(Schuberth et al., 1967; Murrin et al., 1977; Roskoski, 1978) as a Fig. 1.1 Diagramatic representation of the frog neuromuscular junction.

The slender terminal branches of a motor-nerve axon, lacking the myelin sheath of the rest of the fibre, lie in gutter-like depressions

in the membrane of a muscle cell (a). Each terminal region is covered

by a Schwann cell that embraces it with fingerlike processes, sub

dividing it into 300-1000 regularly spaced compartments (b). In the

nerve terminal, the neurotransmitter ACh is distributed between the

cytoplasm and synaptic vesicles. When an impulse arrives at the nerve terminal, ACh is released into the synaptic cleft from vesicular and/or cytoplasmic compartments (discussed in detail later) at an area of pre-

synaptic membrane known as the active zone. The transmitter molecules

then diffuse across the cleft and bind to specific receptors (AChR) embedded in the muscle-cell membrane at the crests of the junctional

folds (c). The binding of ACh to the receptors opens channels in the latter membrane, allowing both Na+ and K+ ions to flow through the membrane. The Na+ influx exceeds the K+ efflux resulting in depolarisation of the post-synaptic membrane; this triggers a large

impulse (an endplate potential) that causes the muscle fibres to contract. ACh is subsequently destroyed by AChE which is distributed throughout the fibrous matrix that fills the synaptic cleft and lines the junctional folds. Here the matrix has been cut away near the active zone to reveal the receptors. (Adapted from Lester, 1977). 7

Fig. 1.1

* MYELIN SHEATH

SCHWANN CELL

* ACETYLCHOLINESTERASE

ACETYLCHOLINE RECEPTOR

JUNCTIONAL FOLD - 8 -

source of substrate for further synthesis of ACh by the enzyme choline

acetyl transferase (CAT) (Hebb and Whittaker, 1958; Whittaker, 1965;

Haga and Noda, 1973). The arrangement of components at the neuro

muscular junction (NMJ) that are thought to be involved in cholinergic

* neurotransmission are illustrated schematically in Fig. 1.1. As

illustrated, the "active zones", thought to be intrinsically involved in

ACh release are found to be situated opposite areas of the postsynaptic

membrane that contain the highest density of AChRs; this close juxta- % position of pre- and post-synaptic sites ensures an efficient relay of

information between the two membranes. Although the events leading up

to release of transmitters have been known for many years, to date, no

* molecular component directly involved in the release process has been

identified.

1.1.2 Modes of neurotransmitter release. Most studies on transmitter * ------release mechanisms have been performed on the cholinergic NMJ; hence,

the following sections will refer mainly to this system although similar

processes may occur in the release of other neurotransmitters, n 1.1.2.1 Quanta! release of transmitter. Katz (1962) suggested

that ACh was released in defined packets which he called quantal

units and that one mepp was the electrophysiological response

# evoked by one quantum of ACh. More recently it has been proposed

that one quantum of ACh gives rise to one sub-mepp and that a mepp

is the response to synchronous release of several quanta (Kriebel

^ et al_., 1976; Kriebel, 1978). However, the actual content of

transmitter in one quantum is still a matter of contention.

Many hypotheses have been put forward for the mechanism by

which transmitters are released in quanta. Quantal release could

occur by exocytosis of transmitter from all or a sub-population of

vesicles, opening of a pore (for a fixed duration of time) or operation of a mechanism in the membrane for discharge of some

preformed store or carrier.

Following the discovery of ACh within isolated synaptic

vesicles (de Robertis et al_., 1962), it was proposed that these

vesicles located within the presynaptic nerve terminal were packed

with the transmitter and that during random movement would touch

and fuse withthe presynaptic membrane at critical contact zones

and discharge their contentsinto the synaptic cleft; nerve

stimulation would increase the probability of vesicle fusion at

the contact zones (Katz, 1962). This series of events merged into

the vesicular hypothesis of transmitter release. However, to date

there is no direct evidence that a given vesicle contains a

quantum of ACh, which on stimulation is released into the synaptic

cleft in the required time limits. In order for vesicular release

to be a viable mechanism it must satisfy several criteria: each

vesicle should contain 1 quantum of ACh giving rise to one mepp; a

relationship should exist between the number of filled vesicles

and the post-synaptic response; exocytosis should occur before the

appearance of mepps; vesicular ACh ought to be released directly

and preferentially; also, compounds free within the vesicle

should also be released. It is now clear that many of these

criteria are not satisfied; synaptic vesicles do not contain CAT

(Fonnum, 1966) or newly-synthesised ACh and thus the latter cannot be released preferentially (Suszkiw et a K , 1978). In addition,

neither vesicular ACh nor the mean number of vesicles were modified by nerve stimulation; up to 50% of the ACh present in

the presynaptic terminal occurs free within the cytoplasm and the exchange of cytoplasmic ACh with vesicles, under resting

conditions, is very slow (Dunant et al_., 1972; Zimmerman and

Whittaker, 1974). Using a false precursor in the synthesis of - 10 -

ACh, Large and Rang (1978) suggest that .transmitter is probably

not stored as independent quanta, but as a continual mixture of

releasable ACh. To circumvent some of these problems, hetero

geneity in the vesicle population was suggested (Zimmerman and

4 Whittaker, 1977); on stimulation, newly synthesised ACh is

preferentially taken up by metabolically-active vesicles in close

apposition to the presynaptic membrane where, owing to the influx

of Cac ions, vesicles are being recycled. However, the half- * life of protein components of presynaptic membranes and vesicles

is about 10 days (von Hungen et a_K, 1968) indicating a degree of

membrane conservation which is inconsistent with a rapid turnover

* of membrane by an exocytotic-endocytotic mechanism; also, such a

mechanism is energetically unfavourable. Therefore, although

there is overwhelming evidence in favour of exocytosis of synaptic

vesicles (Heuser, 1978), there is no direct evidence for its * involvement in transmitter release.

More recently, to account for the available data on the

nature of ACh release, dynamic 'operator1 systems have been * imagined (Israel et alk, 1979; Tauc, 1979). Such systems must be

apposed to (or be part of) the presynaptic membrane and bind ACh

saturably; this saturability would account for the constant

* quantal size despite large fluctuations in the concentration of

cytoplasmic ACh during nerve activity (Dunant et al_., 1977). In

addition, the 'operator' must be charged from the cytoplasmic pool

fc of ACh and release it in a non-electrogenic manner, as the quantal size is independent of the extent of membrane polarisation (Cooke

and Quastel, 1973; Katz and Miledi, 1965b; Simmoneau et al., 2+ 1980); Ca ions would be required as a trigger for such a

system.

In order to describe a mechanism of ACh release that does not directly involve vesicular ACh, Tauc (1.979) has put forward an interesting hypothesis. He proposes that a macromolecular structure is involved which binds ACh with high affinity and is exposed to the cytoplasm; the term "vesigate" was used as such a structure combined properties attributed to vesicles (namely confinement of prepackaged ACh) and a membrane gating mechanism.

Exactly how much membrane space such structures would require and how they cause transmitter release is unclear; it has been suggested that a polymeric macromolecule could be involved.

Israel et a1_. (1979) imagine that a macromolecule, organised like the synaptic heavy form of AChE, could form a triple tetramer and bind 480 ACh molecules, enough to evoke one sub-mepp. Ten of 2 these complexes, (which would occupy less than O.Olum of membrane surface), triggered synchronously, would result in the release of a single quantum of ACh; under optimal condtions it is estimated that cholinergic synapses release about 1 quantum of ACh 2 per pm (Yrensen et_ al_., 1980). However, it has yet to be shown that such a mechanism is compatible with the rapidity of release and the recovery period. It is possible that a contractile system is involved in neurosecretion as proteins similar to actin and myosin have been found in nerve endings (Unsicher et al_., 1978) and presynaptic membranes (Stadler and Tashiro, 1979). Although the rate of contractile protein action is not thought to be rapid enough for vesicle exocytosis, it may allow intramembrane dis placement of several angstroms within 0.1 msec (LIinas and Heuser,

1978) which may be sufficient for activation of a vesigate or another such 'operator' complex. The linking of several vesigates to a single contractile protein may overcome difficulties in achieving synchronous release. When the nerves are in a resting state, spontaneous mepps may result from asynchronous release of Fig. 1.2 Schematic representation of possible mechanisms of ACh release

at the synapse, (adapted from Tauc, 1979)

and eventually binds to triggering and synchronizing molecular structures [TSMS (1)]. If vesigates (2) are saturated by cytoplasmic

ACh, quantal release takes place (situation II). Release does not occur if vesigates are not saturated (situation III) or if calcium did not reach TSMS (situation I). Acetylcholine is also released in a non- quantal manner by release mechanism related (3) or not related (4) to

Ca influx. Low Ca^ concentration in cytoplasm is rapidly + recovered by activation of Ca 2 -buffering, ATP-dependent mechanisms.

These are smooth endoplasmic reticulum (5), mitochondria (6), and particularly synaptic vesicles (7) in which Ca may replace ACh and + ATP. During rest period after moderate stimulation, sequestered Ca2

(8) can be slowly released from vesicle in cytoplasm (9), with vesicle + 2+ thus recovering its initial condition. Ultimately Na -Ca exchange 2+ mechanism (not represented here) in plasmalemma brings the Ca content of terminal to its original level. By contrast, after prolonged or heavy stimulation, vesicle is saturated by Ca , it fuses with plasma membrane and extrudes its contents, essentially Ca2+, by exocytosis (10). - 13 -

ACh from single vesigates or some other channel momentarily

opened. The basis of the vesigate hypothesis, as described by

Tauc (1979), is summarised in Fig. 1.2. However, there is one

problem with this hypothesis; to avoid hyperpolarisation of the

* presynaptic membrane, efflux of ACh would have to be accompanied

by efflux of a counterion, such as chloride ion.

If this vesigate or some other ’operator' mechanism is

functional in the release of ACh, then one needs to explain the * role(s) of synaptic vesicles. It is possible that the vesicles

may act as a reserve store of transmitter, accepting it from the

cytoplasm when release rates are low and replenishing cytoplasmic

pools when rates are high. A more dynamic role for synaptic + . vesicles may be in the sequestration of internalised Ca 2 ions

(Israel et al_., 1980); sequestered Ca^+ ions may be subsequently

released when nerves are at rest and expelled from the cell by the ♦ j . O x Na -Ca exchange mechanism. Only after prolonged stimulation

would Ca -containing synaptic vesicles fuse with the pre- + synaptic membrane and extrude Ca 2 by exocytosis; this may

explain why in normally-stimulated synapses exocytotic pits on the

membrane are hardly ever seen in electron micrographs. A capacity

to buffer cellular Ca concentrations may also explain the

presence of synaptic vesicles at the crayfish electrical synapse

(Perachia, 1973 a, b).

1.1.2.2 Non-quanta! release of transmitter. Non-quantal release

* of ACh results in a small continued depolarisation of the post- synaptic membrane after inhibition of AChE; it is revealed by

suddenly blocking the AChR with d-tubocurarine causing an abrupt

hyperpolarisation, the amplitude of which is presumed to be

proportional to the rate of non-quantal ACh release. Non-quantal

release is thought to comprise a larger proportion of - 14 -

spontaneously released ACh than the quantal form (Katz and Miledi,

1971; Fletcher and Forrester, 1975); there is also evidence that + at least part of this non-quantal release is Ca 2 - dependent and

may be induced by stimulation (Mitchell and Silver, 1963).

Quantal and non-quantal ACh release appear to involve different 4 release mechanisms since they seem to respond differently to nerve

stimulation (Katz and Miledi, 1981), treatment with ouabain,

changes in temperature and Ca or K concentrations (Vizi

4 and Vyskocil, 1979). On the other hand, perhaps the main

difference between quantal and non-quantal release is their graded

capacity for synchronisation of releasing units.

* 1.2 BOTULISM

In an attempt to probe the mechanism(s) of transmitter release,

many researchers have used poisons (i.e. neurotoxins) that appear to

specifically affect various presynaptic processes (discussed in detail

later). For compounds to be poisonous they must be active in minute

quantities, highly selective in their site of action, not readily

metabolised, and impairment of the process(es) on which they act must be

essential to the life of the organism affected. One such group of

compounds are the botulinum neurotoxins, produced by the gram-positive

bacillus Clostridiurn botulinum; the toxin from this anaerobic spore

forming bacteria gives rise to the syndrome known as ’botulism'.

Toxigenic strains of Cl. botulinum may produce one or more of eight

imrnunologically distinguishable neurotoxins (A, B, Cj, C2 , D, E, F,

and G) having similar structure and mode of action, in that they all

inhibit the release of ACh from cholinergic motor-nerve terminals

(reviewed by Simpson, 1981a). These toxins are the most potent neuro

toxic proteins known to man; it is estimated that 0.05-0.5ug of

purified neurotoxin represents one human lethal does (orally taken). - 15 -

The word 'botulism' derivates from the latin word 'botulus'

meaning sausage, this being a common source of the syndrome in the

nineteenth century. The symptoms of botulism are generally evident

within 12-36 hours of infection, firstly in the cranial nerve territory m and then descending; these generally consist of a bilateral weakness

(which may be asymmetrical) with the face, tongue, pharynx, neck and

limbs being most affected (Cherington, 1980). This characteristic

flaccid paralysis of skeletal musculature is rapidly followed by fatal Ml respiratory failure; only in some cases can this be prevented by

artificial ventilation. I here are three ways in which botulism may be

acquired, namely ingestion of contaminated food, infection of wounds or

* in infants, ingestion of spores of Cl. botulinum.

Food botulism is generally due to ingestion of contaminated raw

fruit or vegetables, food inadequately cooked or preserved, with types

A, B and E usually being the infective forms in humans (Feldman et al.,

1981). The toxin is formed by Cl. botulinum on foodstuffs, ingested and

taken up into body fluids by the intestinal mucosa. Although spores of

Cl. botulinum are heat resistant (Ito et al_., 1968), the toxin itself is ♦ extremely heat labile and the majority of cooking procedures rapidly

inactivate it. However, minor outbreaks of botulism still occur,

especially with the advent of home-canning of foodstuffs. In the USA,

* between 1950 and 1979 there were over 800 confirmed cases of botulism

(Feldman et al_., 1981) and in 1978 there was an outbreak in Birmingham

in this country, with several fatalities (P.H.L.S., 19/9; Ball et al.,

% 1982).

Wound botulism is probably the rarest form, but may occur if an

open wound becomes infected with the organism which then produces toxin

and is taken by the bloodstream to exert its paralytic effect. Of such

cases, only types A and B have been documented (Feldman et al_., 1981; cf

Sugiyama, 1980). - 16 -

Of increasing interest in recent years has been infant botulism,

which may now be the most extensive form of this intoxication (Arnon et

al., 1978; Midura and Arnon, 1976; Shield et a K , 1978; Arnon, 1981;

Hatheway et al., 1981; Brown, 1981). Evidence for infant botulism comes

« from the discovery of Cl. botulinum and/or toxin in the faeces of

infants suffering from constipation and neuromuscular weakness. Also,

the 'sudden infant cot death syndrome', where children between 3-26

weeks old may be affected, has been linked in some cases to botulism, M with the occurrence of type A infections slightly more frequent than

type B. Spores of the organism are thought to be the infective form,

which may colonise the intestine and produce the toxin (Midura and

* Arnon, 1976; Arnon, 1980). As the spores are taken orally this may also

be regarded as a type of food poisoning. It is likely that

botulinisation of human infants may have a microbial ecological basis

since adult laboratory animals are not particularly susceptible to

infection by injection with spores, whilst germ-free animals are very

susceptible (Arnon et al_., 1977; Sugiyama, 1981).

* 1.3 TOXINS FROM CL. BOTULINUM

1.3.1 Botulinum toxin-haemagglutinin complexes. Almost a century has

now passed since van Ermergen (1897) originally described botulism as

being due to the presence of a bacterial toxin. In 1946, a neurotoxic

component from cultures of Cl. botulinum type A was isolated in a

crystalline form and was shown to have a relative molecular mass (Mr)

of about 900000 (Lamanna et al_., 1946; Abrams et al_., 1946). This

crystalline toxin was later shown to be a complex of two major protein

components, one of which displayed haemagglutinin activity but no

toxicity and the other which was highly neurotoxic (Mf of 150000) but

devoid of haemagglutinin activity (DasGupta et a1_., 1966). The haemagglutinin component was subsequently shown to exist in three states

of aggregation with M of 290000, 500000 and 900000 (DasGupta and

Boroff, 1968). All eight types of Cl. botulinum can produce neurotoxin

although there is some uncertainty whether or not they all produce

haemagglutinin. Using type C organisms and chicken erythrocytes Lamanna

and Lowenthal (1951) could not detect haemagglutinin activity. However,

Boroff and DasGupta (1971) found that type C organisms produce a

substance capable of agglutinating human erythrocytes, indicating that

haemagglutinins may have some degree of species specificity, as is the

case with botulinal neurotoxins that show differential inter-species and

intra-species specificities (reviewed by Simpson, 1981a; Sugiyama,

1980).

1.3.2 Structure and activity of the neurotoxins. The botulinum neuro

toxins (excepting types C and E toxins) are synthesised as a single

polypeptide chain (Mr ^150000) which may be split by endogenous

proteases to form two dissimilar subunits with Mr of about 100000 and

50000 (DasGupta, 1981). This process is known as ’nicking' and may also

be achieved in vitro by mild trypsinisation; the dichain molecules

produced naturally or with trypsin are indistinguishable (Krysinski and

Sugiyama, 1981). In type E cultures, the toxin is only found in its single chain form suggesting that the nicking protease is absent

(DasGupta and Sugiyama, 1977a); however, trypsinisation yields its dichain form (DasGupta and Sugiyama, 1972a). Type C toxins have not yet been isolated in an unnicked form.

In addition to being synthesised as single chain molecules, the above toxins are only partially active when formed and require an activation step before their full potency is realised (Fig. 1.3). This activation may be performed in vitro using trypsin or in vivo by an endogenous trypsin-like enzyme(s) (TLE); the latter enzyme was so- Fig. 1.3 Generalised structure of botulinum neurotoxins and possible pathways of nicking and activation. (Adapted from DasGupta, 1981)

Botulinum neurotoxin is synthesised as a single chain polypeptide of **,150000 having a low toxicity (left). It is converted to its fully active dichain form (right) by nicking (position 1) and activation

(position 2, represented here as cleavage of a small portion from the tail of the toxin's larger subunit). Tryptic activation cleaves the toxin at positions 1 and 2 (centre route), but in growth cultures activation may arise by sequential nicking and activation, or vice versa

(upper and lower routes, respectively). An endogenous protease (TLE) is believed to cause partial activation without nicking; on nicking, to form two subunits (Mr of 100000 and 50000) linked together by a disulphide bond (except in type C2 toxin), this partially activated toxin becomes fully active. An intramolecular disulphide loop is also thought to exist near the terminal region of the heavier chain. - 19 -

called because when isolated from type B cultures (DasGupta, 1971) it

was found to be an SH-dependent enzyme with substrate specificity

restricted to lysine and arginine bonds (DasGupta and Sugiyama, 1972 b).

However, the bonds cleaved by TLE and trypsin are not entirely

identical; while trypsin caused activation and nicking of type E neuro

toxin, TLE produced only partial activation without nicking (DasGupta

and Sugiyama, 1976). This implies that cultures producing nicked and

activated toxin have two enzymes, one for nicking and another for

activation. When TLE-treated type E toxin (unnicked, partially

activated) was trypsinised it was nicked and further activated.

Evidencefrom type C^ toxin argues in favour of the upper route in

Fig. 1.3 asbeing the normal sequence of events. Ohishi et a K (1980)

isolated Z^ toxin as two separate chains; when a combination of the

two chains (nicked toxin) was trypsinised it was activated. Moreover,

if the light chain was trypsinised separately and then combined with the

heavy chain there was no activation; on the other hand, if the heavy

chain was trypsinised and then combined with the light chain there was

activation. Hence, the fragment cleaved at the end of the heavy chain

in Fig. 1.3 represents the effect of trypsinisation associated with activation.

Another common feature amongst the neurotoxins, with type (^ being the sole exception (Ohishi et al_., 1980), is that their subunits

are connected by inter-chain disulphide bond(s) (DasGupta, 1981). The integrity of disulphide bonds, at least in types A, B and F toxins is

necessary for full expression of neurotoxicity; cleavage with reducing agent results in almost complete loss of the protein's original activity

(Sugiyama et al_., 1973). This loss occurs even upon treatment of un nicked molecules and is most probably due to conformational changes; whether this results primarily from reduction of intramolecular or intermolecular disulphide bond(s) remains to be established. 1.3.3 Bacteriophages and toxigenicity of Cl. botulinum'. The occurrence of non-toxigenic strains of Cl. botulinum, together with the fact that all types of this organism carry plasmids of phages (Dolman and Chang, 1972; Inoue and Iida, 1968; Scott and Duncan, 1978) suggests that production of neurotoxins by Cl. botulinum may be mediated by bacteriophages or plasmids. This would then be analogous to toxin production by Corynebacterium diphtheriae (see below).

Cl. botulinum types C and D may produce types C^, Cg or D toxins (Eklund and Poysky, 1974); type C strains produce mainly C^ toxin with minor amounts of Cg and D toxins whereas type D strains produce predominantly D toxin with minor amounts of C^ and C2 toxins. 'Non-toxigenic' forms of type C strains were obtained after treatment with acridine orange; this resulted in the absence of C1 toxin. If a lysate of the parent culture was added to the non- toxi genic culture, the survivors produced Cj toxin again (Inoue and Iida,

1970). This curing and reinfection was thought to be due to a lysogenising bacteriophage. Later, Inoue and Iida (1971) reinfected a cured type C strain with bacteriophage from a toxigenic type D culture, resulting in the production of type D toxin. Moreover, elimination of the tox+ phage for the dominant toxin in types C and D strains gave cured strains that did not produce one of the other minor toxins (i.e.

C^ of type D strains or D of type C cultures) (Eklund and Poysky,

1974). This suggests that one phage may control the production of two types of toxin. However, the other minor toxin, C2 , in type C and D cultures does not appear to be phage-mediated (Eklund and Poysky, 1972).

Inter-species conversion of strains of Clostridia as described above

(also see Eklund et al_., 1974) raises a problem of nomenclature; pathogenic strains of Clostridia are generally classified according to the immunological properties of the toxin they produce. A more accurate classification of species from the genus Clostridium ought to take into - 21 -

account other properties such as DNA-homology and sensitivity to

phages.

1.4 THE ACTION OF BOTULINUM TOXINS *

1.4.1 Interaction with neuronal tissues. Original attempts to localise

the binding of botulinum toxin at the NMJ employed fluorescent- or

ferritin-labelled derivatives of impure toxin-haemagglutinin complexes * (Zacks et al^., 1962, 1968); however, in these studies, no control

experiments to reveal the extent of non-specific binding were performed.

The inherentcomplications when interpreting data obtained from * experiments in which toxin-haemagglutinin complexes were used, are well

illustrated by the fact that tritiated neurotoxin-haemagglutinin complex

was found to bind to neuronal and non-neuronal membranes; the majority

of this binding was inhibited by galactose, an inhibitor of

haemagglutinin (Dolly et al_., 1981). Habermann (1974) and Wiegand et

al. (1976) attempted to monitor the fate of botulinum neurotoxin

following intramuscular injection of a I-labelled derivative * obtained by gel filtration of radiolabel led toxin-haemagglutinin

complex; owing to the low specific radioactivity of such preparations

they were found to be of limited use. This may be the reason why * Habermann (1974) failed to localise any binding sites for the 125 I-labelled toxin at the rat neuromuscular junction. Soon after- 125 wards it was reported that a I-labelled derivative of botulinum

% neurotoxin bound specifically at the murine NMJ (Hirokawa and Kitamura, IOC 1975). However, this group's preparation of I-labelled neurotoxin,

and that used in a later study (Kitamura, 1976), was apparently of low

specific radioactivity (45-150 Ci/mmol) and unstable upon storage

(usable for only a few days). Membrane preparations from tissue of the

central nervous system have been used as a more convenient model for biochemical analysis of neuronal binding site(s) for botulinum toxins.

Immunocytochemical localisation of neurotoxin bound to saturable sites

on brain membranes has been demonstrated (Hirokawa and Kitamura, 1979),

although the sites observed were not located solely at synaptic regions

of the nerve membrane. To date, detailed biochemical studies on the

binding sites for botulinum toxins in brain membranes using radio-

labelled toxin have been restricted; this is primarily due to the low

specific radioactivity (Habermann, 19/4; Hirokawa and Kitamura, 1975;

Kitamura, 1976; Kozaki and Sakaguchi, 1982) and instability (Kitamura,

1976) of the toxin derivatives used or the low affinity of their inter

actions with the receptors (Kozaki, 1979; Kozaki and Sakaguchi, 1982).

It is clear that before any significant advances can be made in the

characterisation of interactions between radio!abelled botulinum toxins

and their specific receptor(s), several criteria must be met. In the

first instance, the neurotoxin must be purified to homogeneity from

haemagglutinin and/or other non-toxic protein contaminants; this will

allow unambiguous observations to be made on the specific binding of

neurotoxin to its receptor(s). I he pure neurotoxin must be subsequently

radiolabelled to high specific activity without significant losses in

neurotoxicity, thus enabling the sensitive detection of small amounts of

this very potent toxin. In addition, the usefulnes of such a

preparation must not be limited by its instability upon storage;

therefore, it must remain active for weeks rather than a few days (as was the case with some of the aforementioned preparations).

The presence of a lag phase in the action of botulinum toxins, electro-physiological findings (discussed in Section 1.4.2) and the known strategy of action for many other microbiol and plant toxins (see

Section 1.5), suggests that toxin uptake may be essential for the toxicity of these proteins. Botulinum neurotoxins (Mr <>.150000) are too large to pass through the membrane via any of the known ion- channels, thus suggesting an active mechanism of uptake, mediated by either itself or a membrane process, such as endocytosis. The time to paralysis in nerve-muscle preparations treated with botulinum toxin may be shortened by increasing the extent of indirect stimulation (Hughes and Whaler, 1962). Owing to the fact that there is a significant correlation between exocytosis and endocytosis (Heuser and Reese, 1973;

Ceccarelli et al_., 1973; Teichberg et al_., 1975) and that endocytosis is an almost universal cell process, the latter mechanism is a likely candidate as mediator of internalisation. Direct evidence that botulinum neurotoxin may be taken up into nerve terminals in an energy- dependent process has now been reported (Black et al_., 1983; Dolly et al., 1984a); whether or not these internalised toxin molecules are effective in blockade of transmitter release has yet to be confirmed.

It has been suggested (Habermann, 1974; Vliegand et al_., 1976) that once toxin has entered the nerve ending it may ascend to the spinal cord by retrograde axonal transport in a similar manner to that which has been ascribed to tetanus toxin (Dimpfel and Habermann, 1973; Habermann et al., 1973; Seib et al_., 1973; Erdmann et al., 1975). By analogy with other bacterial toxins it is possible that after internalisation the toxin molecule is activated by some intracellular process (e.g. within lysosomes) and that this ‘active’ form of the toxin may directly affect transmitter release.

1.4.2 Pharmacological actions of botulinum toxins. Burgen et al_.

(1949) first reported that the site of action for botulinum toxin was the cholinergic nerve terminal, where it was found to inhibit the release of the ACh. Indirect stimulation (via the nerve) of botulinum toxin-treated neuromuscular junction preparations results in a pro gressive diminution of muscular contraction and epp amplitudes;

paralysis occurs at a time when nerve stimulation no longer gives rise

to an epp large enough to depolarise the postsynaptic membrane (Burgen et al., 1949). The toxin binds with apparent irreversibility to rat phrenic-nerve hemidiaphragm well before the onset of paralysis which

suggests that the physical act of toxin binding to its membraneous site may not actually cause toxicity 'per se' but that another process(es) may directly effect toxicity. Following Burgen's work, Hughes and

Whaler (1962) found that there was a correlation between the actual time to paralysis, using a given toxin concentration, and nerve stimulation; the time to paralysis decreased with increasing stimulation. It was later proposed that excitation-secretion coupling influenced the onset of toxicity rather than the depolarisation-repolarisation cycle

(Simpson, 1971, 1973). Anti-botulinum toxin antibody was shown to protect nerve-muscle preparations from paralysis but was unable to reverse the toxin's effects after the onset of paralysis (Simpson,

1974). To bridge the gap between toxin binding to its cell 'receptor' and the onset of paralysis, Simpson (1980) suggested an intermediary step of translocation (internalisation) of the toxin molecule (or part of it) across the membrane to an antitoxin insensitive site. Hence

Simpson's proposed three-phase hypothesis of botulinum toxin's action is: (1) binding of the toxin to its cell surface receptor(s), which is 2+ not temperature dependent or sensitive to Ca concentration; (2) internalisation of the toxin to an antibody insensitive environment , a 2+ process that is highly temperature-dependent and sensitive to Ca and

(3) an, as yet, undefined 'lytic step*, which directly results in the inhibition of transmitter release.

Studies on the action of impure type D toxin when applied in vitro to the frog NMJ showed that the toxin had no effect on propagation of the impulse along nerve fibres or the flow of this impulse into cholinergic nerve terminals (Harris and Miledi, 1971). After botul inisation of these terminals there is no decrease in the postsynaptic responses to ACh or other agonists. Therefore, either the metabolism, of ACh or the excitation-secretion coupling is affected at cholinergic synapses; the toxin does not apparently interfere directly with the metabolism of ACh (reviewed by Gundersen, 1980).

To date, no central action of botulinum neurotoxins, in vivo, has been demonstrated. Previous reports of a central action in vivo employed the use of crude toxln-haemagglutinin complexes (Polley et al.,

1965; Wiegand and Wellhoner, 1977); however, these observations are thought to be due to artifacts of the toxin preparation used, ihere may be many reasons why the purified neurotoxin is inactive at these synapses in vivo; it may not cross the blood-brain barrier, the size of the synapse may be prohibitively narrow, the receptor(s) may be absent or central synapses may be lacking in some other mechanism necessary for expression of the toxin’s activity. One of these possibilities, namely the absence of a receptor is unlikely; specific binding components for botulinum neurotoxin types A, B and E in rat brain preparations (i£ vitro) have been reported (Habermann, 1974; Kozaki, 1979; Kozaki and

Sakaguchi, 1982). In addition, in vitro studies on the central nervous system have shown a decrease in evoked and spontaneous release of ACh from toxin-treated brain synaptosome preparations (Bigalke et al_., 1981;

Dolly et al_., 1982). Moreover, the toxin does not appear to directly affect choline uptake (Bigalke et al^., 1978; Gundersen and Howard,

1978; Wonnacott, 1980b), acetylation of choline by inhibition of CAT

(Wonnacott and Marchbanks, 1976) or levels of endogenous ACh (Gundersen and Howard, 1978) as measured in rat brain synaptosome preparations.

Botulinum neurotoxin has also been shown to have a lesser effect on the release of other neurotransmitters, such as glycine, 4-amino- butyrate and noradrenaline, from synaptosome preparations (Bigalke et - 26 - al., 1981). It is generally believed that botulinum toxin, at least in the peripheral nervous system, is specific for cholinergic synapses, although early reports indicated an ability of toxin complexes to act presynaptically to inhibit noradrenaline release in the adrenergically innervated mouse vas deferens (Whaler, 1967; Westwood and Whaler, 1968;

Holman and Spitzer, 1973). However, these effects may result from uptake of the toxin into the nerve terminal by a low-affinity, non specific mechanism since high concentrations of toxin were used in these experiments; this casts doubt on the occurrence of specific receptors for botulinum neurotoxins at adrenergic synapses. In addition, Dolly et al.(1984a) were unable to show any binding of I-labelled derivative of type A BoNT to adrenergic nerve terminals of the mouse vas deferens; similarly, Mackenzie et al_. (1982) found no electrophysiological effects of BoNT on transmitter release at peripheral adrenergic synapses. However, the apparent inhibition of noradrenaline release by toxin complexessuggests that intra-terminal release of ACh and noradrenaline may be effected by some common or similar mechanism.

Boroff et al_.(1974) suggested that the pharmacological action of botulinum toxin was to inhibit the filling of synaptic vesicles with

ACh, where the observed effect on mepp amplitude would reflect poorly filled vesicles. This idea was put in doubt when it was reported that the distribution of the ACh between vesicular and cytoplasmic pools was unaffected in toxin-treated synaptosome preparations (Wonnacott and

Marchbanks, 1976). Black and brown widow spider venoms (B1WSV and

BrWSY) act by facilitating fusion of synaptic vesicles with the presynaptic membrane giving rise to a large increase in the frequency of mepps (Longeneckeret al_., 1970). When these venoms act on botulinised neuromuscular junctions, the burst of mepps is similar to that found in control preparations (Pumplin and del Castillo, 1975; Cull-Candy et al_.,

1976a; Kao et a K , 1976; Pumplin and Reese, 1977). This suggests that botulinum toxin does not affect the quantal content of the individual

synaptic vesicles. Owing to possible heterogeneity in the population of synaptic vesicles, it has been hypothesised that botulinum toxin interferes with the packaging of ACh into those vesicles that are the usual source of transmitter for normal, indirectly stimulated release.

This would therefore result in a diminished quantal release in the mepps and may account for the skewness in amplitude distribution (Pumplin and del Castillo, 1975). The possibility of heterogeneous populations of vesicles must therefore be borne in mind when considering the idea of impaired vesicle filling (Harris and Miledi, 1971; Pumplin and del

Castillo, 1975). Another hypothesis proposed by Kriebel et a K (1976), is that each vesicle contains one sub-mepp and that a mepp results from synchronous release of several sub-mepps. In this case, low amplitude mepps would be observed if release of ACh was from fewer vesicles. All this besides, taking into consideration the vast number of cholinergic nerve terminals present in an animal and the excess of synaptic vesicles contained within them, the concept that botulinum toxin has a direct action on these vesicles appears incompatible with the toxin's unique potency.

The effect of botulinum toxin on transmitter release may be a com posite one involving more than one site of action. The action of the toxin may be dissected into separate entities; the blocking of post- synaptic responses due to presynaptic nerve stimulation as measured by muscle twitch or decrease in epp amplitudes, a decrease in spontaneous mepp frequency and a positive skew in the distribution of mepp ampli tudes in paralysed nerve-muscle preparations (Boroff et al_., 1974; Cull-

Candy et al_., 1976a; Harris and Miledi., 1971; Dolly et al_., 1982). The time to paralysis in toxin-treated preparations is found to be prolonged when buffer containing low Ca^+ or high Mg^+ (a Ca^+ antagonist) concentrations were used (Simpson, 1973) which poses the question as to whether transmitter release is necessary for paralysis to 2+ occur. Furthermore, high Ca concentrations confer a slight improvement on indirectly stimulated ACh release from toxin-treated 2+ preparations; Ca ionophores or tetraethyl ammonium (both of which increase neuronal Ca concentration) also increased ACh release from partially paralysed muscles. This indicates that the transmitter release mechanism has not been destroyed, but requires an abnormally 2+ high cytoplasmic Ca concentration to be functional (Cull-Candy et al., 1976b; Simpson, 1978). Whether or not botulinum toxin affects 2+ membrane transport of Ca has been investigated by many groups with differing conclusions. Wonnacott and Marchbanks (1978) failed to show any effect on Ca flux in toxin-treated synaptosome preparations under resting or K+-stimulated conditions; on the other hand, Hirokawa and Heuser (1981), using indirect morphological studies at the neuro- 2+ muscular junction, reported that the toxin may block the Ca -channels in the plasma membrane. A more recent and direct study, using external electrodes to record presynaptic membrane currents at motor endplates, 2+ showed that the inward Ca current was unaffected by botulinum toxin

(Dreyer et al_., 1983). With regards to the LD^q for botulinum neuro- g 2+ toxin (in the order of 10 molecules) and the number of Ca - channels present in nerve membranes, it is doubtful whether the toxin interacts directly with membrane Ca -channels (Hanig and Lamanna,

1979). Additional evidence against a direct effect of the toxin on 2+ Ca -channels comes from the action of batrachotoxin, an irreversible activator of sodium channels (Albuquerque and Daly, 1977), on botulinised preparations. 8atrachotoxin, a steroidal alkaloid, causes the release of a barrage of mepps from the neuromuscular junction; this - 29 -

Fig. 1,4 Schematic diagram of a nerve terminal illustrating the two

types of vesicular transmitter release (I and II) as proposed by

Thesleff. (From Thesleff, 1981)

• f t

♦ 2+ 2+ process is Ca -dependent and may utilise Ca already present in the nerve endings. This toxin does not show the same release of mepps from botulinum-treated tissue and therefore botulinum toxin probably 2+ prevents the physical release of ACh after internalisation of Ca 2+ rather than an effect on Ca -influx (Simpson, 1978). Also, Lundh et al. (1977) proposed that botulinum toxin may cause the nerve terminal 2+ to become less responsive to extracellular Ca concentration by 2+ lowering the affinity for or efficacy of Ca at some intracellular site.

Electron-microscopy has revealed some details of sites thought to be involved in the presynaptic release of transmitters. Exocytosis, the proposed normal mechanism for ACh release, appears in freeze-fracture electron micrographs as deformations in the presynaptic membrane. In botulinised preparations these deformations are almost entirely absent

(Pumplin and Reese, 1977). Neurotransmitter release sites are thought to be concentrated on the presynaptic membrane opposite areas of post- synaptic membrane containing a high density of ACh receptors (Heuser et al., 1974). Such concentrations of release sites are known as ‘active zones* and appear as ridges bordered by processes that may extend through the membrane (Jones, 1975). As mentioned above, BrWSV causes release of ACh from botulinised membranes, but not very efficiently however, from the active zones. In accordance with this, Kao et al.

(1976) reported the presence of “log-jams" of synaptic vesicles at the active zones in botulinum-treated tissues. These observations suggest that botulinum toxin acts at, or near, the active zones to regulate nerve-stimulated exocytosis and release of ACh. An extension of these findings has led Thesleff (1981) to hypothesise the existence of two kinds of vesicular transmitter release (see Fig. 1.4). The first (I) is from the active zones, which gives rise to the normal Gaussian distribution of mepp amplitudes. Release from these sites is thought to 2+ be quantitatively related to intracellular Ca concentration,

Ca -flux and nerve-terminal depolarisation. The mepps and epps resulting from this type of release have rapid and uniform rise times owing to the close proximity of these sites to the high density of post- synaptic AChRs. In addition, a second mechanism of quanta! vesicular transmitter release is hypothesised at nerve terminals (site II, see

Fig. 1.4). The sites involved are independent of intracellular 2+ 2+ Ca -concentration and Ca -flux and thus not affected by nerve stimulation and depolarisation. Unlike the active zones, this second population of sites (II) are thought to be widely dispersed in the nerve terminal and therefore give rise to mepps with a variable and generally prolonged rise time. It is proposed that botulinum toxin blocks the release of ACh from type I sites but not from type II sites, which as well as explaining the block of stimulated ACh release by nerve depolarisation may explain the decrease in frequency and skewed distribution of mepps in poisoned neuromuscular junctions.

At present no details are known of botulinum toxin's interaction with any cellular component directly involved with transmitter release although various models have been put forward. A ‘pipe and valve1 hypothesis has been proposed by Lamanna (1976) which suggests that when a ‘valve1 opens to enable presynaptic release of ACh, botulinum toxin acts as a mechanical 'plug' preventing further transmitter release.

This and other 'one-hit' mechanisms (such as an effect on Ca - channels already discussed) suffer the drawback that only very few toxin molecules are required to have a lethal effect; since the numbers of any component involved in synaptic transmission so far reported

(Ceccarelli et al_., 1973; Ceccarelli and Hurl but, 1975) greatly exceed this number of toxin molecules, it is doubtful whether this type of mechanism is responsible for the action of botulinum toxin. - 32 -

The model described above for the action of botulinum toxin on quanta! release of ACh (Thesleff, 1981) may also be reconcilable with an

'operator' system for non-vesicular release (as detailed earlier,

Section 1.1.2.1). Sites I and II may represent identical 'carriers' of

ACh with synchronisation of function being the significant difference between them (other than their distribution; i.e. sites I restricted to the active zones and sites II being randomly distributed throughout the presynaptic nerve membrane). Release of ACh may be synchronised by a contractile system which is dependenton critical concentrations of

Ca for its action (as in muscle contraction); this contractile system would only be present in juxtaposition to the active zones and 2+ would have a high affinity for Ca ions in its resting state.

Botulinum toxins would thus interfere either directly or indirectly with the contractile mechanism such that it would be locked in a conformation having a low affinity for Ca ions; hence very high intracellular 2+ Ca ion concentrations would be required to effect subsequent release from these sites. Type II sites, in the absence of such a system, would be unaffected by botulinum neurotoxin and continue to release quantal amounts of ACh asynchronously. This type of model has one main advantage over that of Thesleff (1981) in that it does not require heterogeneous populations of quantal release sites; it can also be envisaged how a small number of molecules may prevent ACh release from the active zones. If botulinum neurotoxins exert some multiplicative

(e.g. enzymic, catalytic) activity in blocking synchronous release at the active zones, this may easily explain the unique potency of these toxins. This is not a new concept since other microbial toxins, e.g. cholera and diphtheria toxins (van Heyningen, 1982a; Collier, 1975) and plant toxins, e.g. abrin ad ricin (Olsnes and Pihi, 1977), have been shown to have some enzymic function (discussed below). - 33 -

1.5 OTHER BACTERIAL AND PLANT TOXINS.

Bearing in mind the aforementioned characteristics of the

structure of botulinum neurotoxins, it is pertinent here to mention * analogies with other bacterial and some plant toxins (see van Heyningen,

1982b).

1.5.1 Cholera toxin. Cholera toxin is an oligomeric protein (Mf * %84500) secreted by Vibrio cholerae. It consists of two heterologous

subunits, A and B, linked by non-covalent forces in a stoichiometric

arrangement of AB^ (van Heyningen, 1982a). The B subunits (Mr 4 ^11500) are arranged in a ring whilst the A subunit comprises two poly

peptides, A^ (Mr = 22000) and A2 (Mr * 5000), linked by a di

sulphide bond (reviewed by van Heyningen, 1977, 1982a). Subunit B has

M been shown to bind to cells via the monosialoganglioside GM^

(Cuatrecasas, 1973a,b,c,d; Holmgren et a K , 1973; King and van

Heyningen, 1973) but otherwise has no activity. It is interesting to

t note that V. cholerae secretes a neuraminidase, an enzyme that cleaves sialic acid residues from polysialogangliosides to give GM^ and other

lower gangliosides. The enzyme may therefore play a pathological role

during infection by V. cholerae, in increasing the number of toxin ♦ receptors on the cell membrane (King and van Heyningen, 1973). The

binding of subunit B to GM^ is a prerequisite for the action of this

toxin and is thought to somehow facilitate penetration of the cata-

% lytically active A^ polypeptide into the membrane; the mechanism of

entry is not presently understood. Van Heyningen (1982c) reported that

binding of subunit B to GM^ caused a conformational change in subunit

A, thereby possibly allowing more direct access of the A^ fragment to

the membrane. It has also been suggested that external reduction of the

disulphide bond connecting the A^ to the polypeptide occurs in order to allow the peptide to penetrate through the membrane

(Tomasi and Montecucco, 1981); this reduction may be facilitated by thiol protein disulphide exchange at external surface of the membrane.

In addition, binding of cholera toxin to cell membranes may cause clustering of membrane gangliosides (and possibly proteins) and, if these clusters arrange to form micellar structures, this may allow the passage of the hydrophobic A^ fragment through the membrane (Tomasi et al., 1982). There is a lag time in the action of this toxin which is thought to represent the time taken for the A1 polypeptide to traverse the membrane to its site of action (adenylate cyclase) on the inner membrane surface. It is now known that cholera toxin acts by maintaining adenylate cyclase in its active form by inhibiting its

GTPase activity; the GTPase of adenylate cyclase hydrolyses GTP to GDP in the conversion of the active cyclase to an inactive form. The chemical reaction involved is the ADP-ribosylation of the GTPase which is catalysed by the A^ polypeptide of cholera toxin. The resultant effect is a large increase in cyclic AMP leading to massive diarrhoea and possibly death through dehydration. More detailed descriptions of the action of cholera toxin may be found elsewhere (Johnson, 1982; van

Heyningen, 1977, 1982a).

1.5.2 Diphtheria toxin. The bacterial toxin whose mode of action is most fully defined at present is diphtheria toxin from Corynebacterium diphtheriae. It is a protein of Mp 62000, which is synthesised and secreted as a single polypeptide being subsequently nicked by proteases to form a dichain molecule linked together by a disulphide bond

(reviewed by Collier, 1975; Pappenheimer, 1977; Uchida, 1982). It is interesting that, as in the case of at least some botulinum neurotoxins, production of diphtheria toxin is also related to infection of bacilli by a lysogenising bacteriophage (Pappenheimer, 1977; Uchida, 1982). The subunits of nicked diphtheria toxin are known as fragment A and fragment

B with Mr of 24000 and 33000 respectively. It is believed that fragment 3 is responsible for binding of the toxin molecule to a membraneous glycoprotein receptor (Proia et al_., 1979) while fragment A is found to inhibit protein synthesis in cell-free extracts (Strauss and

Hendee, 1959). Limited work has been carried out on the binding activity of fragment B due to its instability and hydrophobic nature and is thus not well documented. There exists a lag phase during the action of diphtheria toxin (also seen with cholera, botulinum and tetanus toxins) between the time of binding and the onset of inhibition of protein synthesis; this lag phase is present even when high concentrations (lOnM) of toxin are used (cf. Collier, 1975). The length of the lag time appears to be related to the sensitivity of individual cells to diphtheria toxin. Donovan et al_. (1982) have suggested that the toxin is anchored to cell membranes by interaction with phos- phoinositides on the opposite face of the membrane to which the toxin binds; this may allow the formation of a membrane channel and insertion of the toxin (or part of it) into the cytoplasm. A recent study, using photolabelling techniques, suggests that both subunits of diphtheria toxin may penetrate into the lipid domains of the membrane (Wisnieski and Zalman, 1983). Toxin may also be internalised by adsorptive endocytosis which may lead to intracellular processing of the protein before its release into the cytoplasm to exert its toxic effect. Evidence for such a route was obtained with chloroquine, a lysosomotropic agent; this compound, which inhibited the cytotoxic action of diphtheria toxin, was shown to prevent lysosomal processing of the toxin without affecting its rate or extent of uptake into mouse kidney cells (Leppla et al.,

1980). It is possible that the toxin may gain access to the cytoplasm by direct penetration of the membrane from an acidic intracellular compartment (e.g. lysosome, endosome). Sandvig and Olsnes (1980) have previously shown that diphtheria toxin may penetrate directly through the surface membrane of kidney cells at low pH. Once internalised to the cytoplasm, fragment A is thought to act enzymically (rather than as an enzyme cofactor) to inhibit protein synthesis by catalysing the ADP- ribosylation of elongation-factor 2 (EF-2), thus preventing trans location of amino-acids from aminoacyl tRNA to newly formed polypeptides

(Collier, 1967; Honjo et al_., 1968, 1971). As EF-2 is not the rate- limiting factor involved in protein synthesis, it is found to be ADP- ribosylated in toxin treated cells long before the end of the toxin's lag phase. Only when EF-2 concentrations are depleted by toxin in activation to low levels, does EF-2 become rate limiting and thus exert its inhibitory effect on protein synthesis (Gill and Dinius, 1973).

Evidence for the correlation between inhibition of protein synthesis and toxicity in vivo comes from studies on cross-reactive forms (CRM) of the toxin produced by mutant strains of C. diphtheriae; these CRMs contain amino-acid substitutions which result in altered enzymic activity and toxicity. CRM 197 is found to be devoid of enzymic activity and non toxic, whereas CRM 176 has a low degree of enzymic activity and is only partially toxic (Gill et al_., 1973; Pappenheimer and Gill, 1973).

Unless diphtheria toxin has a second more important activity, affected in a similar manner by these mutant toxins, then toxicity must be related to ADP-ribosylation. Although some structure-activity relation ships of diphtheria toxin are obviously comparable to those of botulinum neurotoxins, similarities between the latter toxin and tetanus toxin are much more pronounced.

1.5.3 Tetanus toxin. Tetanus toxin (a neurotoxin) is produced by another gram-positive clostridial species, Cl. tetani, as an unnicked

(single-chain) molecule (Mr *>.150000). It may be nicked by endogenous protease(s) to form a dichain molecule with its subunits (Mf of 100000 and 50000) linked by a disulphide bond (reviewed by Mellanby and Green,

1981; Wellhoner, 1982). The subunits are found to be antigenically dissimilar (Matsuda and Yoneda, 1975). Tetanus and botulinum neuro toxins both contain a disulphide loop on their larger polypeptide and may be proteolytically cleaved at a similar position in this subunit to give two polypeptides; one peptide represents half of the heavy chain whilst the other contains the light subunit linked by a disulphide bond to the other part of the heavy chain (cf. Sugiyama, 1980). In addition to the gross structural similarities between tetanus and botulinum toxins, these proteins also have remarkably similar pharmacological actions, despite the striking contrast of spastic paralysis in tetanus and the flaccid paralysis of botulism. Tetanus toxin preferentially acts upon the central nervous system whilst botulinum neurotoxins, in vivo, appear to act solely in the periphery at the neuromuscular junction. It is reported that the observed differences in action of these toxins, at least in vitro, are quantitative rather than qualitative (Habermann, 1981). Despite the many similarities in pharmacological actions, described by Habermann (1981), there is also evidence that tetanus and botulinum neurotoxins may act at different sites of the depolarisation- transmitter release process (Dreyer and

Schmitt, 1981).

A three-step model for the action of tetanus toxin (i.e. binding internalisation and a lytic step) has been proposed (Schmitt et al.,

1981) similar to that for botulinum neurotoxin (Simpson, 1980). Schmitt et al. (1981) suggest the involvement of a phase transition rather than an enzymic role in the toxin’s action, once internalised. Tetanus toxin has been shown to interact with neuronal (Habermann, 1973; Habermann et al., 1980a; Rogers and Snyder, 1981; Yavin et aj_., 1983; Zimmerman and

Piffaretti, 1977) and non-neuronal (Ledley et al_., 1977) tissues. The fact that tetanus toxin interacts through its heavier subunit with di- or higher-sialogangliosides has been known for several years now

(He!ting et a K , 1977; van Heyningen, 1976). Neuroblastoma cells in growth and differentiating cultures were capable of binding tetanus toxin, although only in the latter cultures did tetanus toxin exert any physiological effects (e.g. shortening of processes and diminished adherence (Zimmerman and Piffaretti, 1977). Pretreatment of cells from growth cultures with neuraminidase or 8-galactosidase eliminated subsequent binding of toxin; this inhibition of binding was not observed following treatment of cells from differentiating cultures. From these data, Zimmerman and Piffaretti (1977) suggest that in differentiating cultures, gangliosides are not solely responsible for the 'effective' binding they observed. The binding of botulinum neurotoxin to gangliosides has been reported (Kitamura et a K , 1980) but the physiological significance of these findings need to be ascertained, as incorporation of gangliosides into brain synaptosomal membranes did not significantly affect their sensitivity to the toxin's action on transmitter release (Wonnacott, 1980a).

1.5.4 Abrin and ricin. The potent plant toxins abrin and ricin occur in the seeds of Abrus precatorius and Ricinus communis respectively; their toxicity is due to an inhibition of protein synthesis. Both toxins have an Mr of about 65000 and consist of two subunits (A and B) of similar sizes linked by a disulphide bond (reviewed by Olsnes and Pi hi, 1977,

1982). Reduction of the disulphide bond caused inactivation of these toxins if subsequently administered to animals (in vivo) or intact cells

(in vitro); however, such reduction had an activating effect on the toxin in cell-free systems (Qlsnes and Pihl, 1982). The presence of free sulphydryl groups is not considered important in the action of these toxins in cell-free systems as reduction and alkylation had no effect on their toxicity. Only subunits B bind to specific receptors on the cell surface, interacting with terminal galactose residues, lactose, other galactose- containing polysaccharides, ganglioside GM^ and possibly specific glycoproteins. The A-chains are found to inhibit protein synthesis by inactivation of the 60S ribosomal subunit; both the initiation and elongation of peptide chains appear to be affected (cf. Olsnes and Pi hi,

1982). Inhibition of protein synthesis only occurs when the A-subunit is in a free state; binding of the B-subunit prevents the inhibitory action of the A-subunit. Therefore, the A-chain must be liberated from the B-subunit before inhibition of protein synthesis can occur (Olsnes et al_., 1976). Photolabelling studies indicate that the individual sub units of ricin, especially the A-subunit which has extensive hydrophobic domains, may penetrate the membrane bilayer more efficiently than the intact toxin (Ishida et al_., 1983); thus it may be advantageous for the action of this toxin if the disulphide bond is cleaved prior to membrane penetration. The lag time in the action of abrin and ricin is not observed in cell-free protein-synthesising systems which suggests the rate-limiting step is that of toxin internalisation. Endocytosis is thought to be involved in the uptake of ricin and abrin from the membrane (Sandvig and Olsnes, 1982a); release of endocytosed toxin from intracellular compartments to the cytoplasm is reported to be dependent 2+ on specific Ca ion gradients (Sandvig and Olsnes, 1982b). However, in contrast to the protection afforded by chloroquine and NH^Cl on the action of diphtheria (Leppla et a U , 1980) and botulinum (Simpson, 1982) toxins, these agents slightly sensitised cells to abrin and ricin.

It is interesting that the plants producing abrin and ricin also synthesise agglutinin components; perhaps somewhat analagous to haemagglutinin proteins produced by Cl. botulinum. Abrus and ricinus agglutinins exist as dimers, held together by non-covalent forces; each protein is comprised of two subunits (A and B) linked by a disulphide bond. The 3-subunit has the binding capacity of the protein whilst the

A-subunit inhibits protein synthesis, although much less efficiently

than the respective subunits of abrin and ricin (Olsnes andPihl,

1982).

1.6 PRESYNAPTICALLY ACTIVE SNAKE VENOM TOXINS.

Having looked at bacterial and plant toxins that have somewhat

analogous structures to that of botulinum neurotoxins, we turn our

attention to snake venom toxins that are active at presynaptic nerve

membranes.

1.6.1 Snake toxins exhibiting phospholipase Aq activity. These neuro

toxic proteins (e.g. B-bungarotoxin, crotoxin, notexin and taipoxin)

contain one or more polypeptides exhibiting a Ca -dependent phos pholipase (PLAg) activity. Intermolecular disulphide bonds may link these peptides to one or more subunits with no known enzymic activity (Lee, 1972 and 1979; Howard and Gundersen, 1980). As observed with botulinum neurotoxins, reduction of inter- and/or intra-molecular disulphide bonds results in the loss of neurotoxicity (Abe et al_., 1977;

Howard and Troug, 1977). Of the toxins listed above, B-bungarotoxin

(B-BuTx) is presently the most characterised. This toxin consists of two subunits linked by disulphide bridges; the larger A-subunit (Mr =

13000) exhibits PLA2 activity whilst the smaller 3-subunit (Mr 3

7000) is devoid of such enzyme activity (Kondo et al_., 1978). 8-BuTx acts preferentially at the neuromuscular junction where it prevents ACh release from motor nerve terminals (Chang et , 1973; Abe et al.,

1977; Thesleff, 1977); it causes an initial decrease in epp amplitudes at motor endplates followed by a transient increase that precedes blockade (Chang and Huang, 1974; Abe et al_., 1977; Caratsch et al., 1981). Choline uptake is also reported to be inhibited, at least partially, by 8-BuTx; however, this may be due to a secondary effect of the toxin's action (Dowdall et al_., 1977). Like botulinum toxin, B-BuTX does not affect propogation of the action potential along the nerve, CAT activity or deplete the quanta! content of synaptic vesicles (Abe et al., 1977). Crotoxin, notexin and taipoxin also have a similar pattern of effects on transmitter release at the neuromuscular junction but are thought to act at different sites (Cull-Candy et al_., 1976b; Chang et al., 1977; Chang and Su, 1980; Gundersen and Jenden, 1981). In addition, with the exception of B- BuTx, they are all known to have some postsynaptic activity (Chang, 1979; Bon et al_., 1979).

Non-toxic PLAg (Volwerk et al_., 1974) and neurotoxic phos pholipases (Halpert et al_., 1976; Howard and Troug, 1977; Abe et al.,

1977) may be inactivated by chemical modification of a single histidine residue at their active site. Following histidine-modification of

8-3uTx only the small original decrease in epp amplitude or mepp frequency was seen which suggests that the second and third phases of its action are due to its PLAg activity (Abe et al_., 1976, 1977). In addition, removal of Ca and its replacement with Sr (an inhibitor of PLAg activity) decreases the toxicity of B-BuTx (Caratsch et al_., 1981); under these conditions only the first phase in the toxin’s action is observed, as seen above with chemical modification.

In addition to its peripheral effects, 3-BuTx also prevents release of neurotransmitters from brain synaptosomes (Spokes and Dolly,

1980; Tse et al_., 1980; Wernicke et al_., 1974) and neurotransmission

(measured electrophysiologically) in brain slices (Dolly et al_., 1980).

These actions are decreased but not abolished in the absence of the toxin's PLAg activity (Dolly et al_., 1980). Neurotoxic PLA2 's, when tested in their ability to attack synaptosomes, mitochondria and myelin, showed a preference for synaptosomes as a substrate. If phospholipids - 42 -

were extracted from these fractions and used as substrates, neurotoxic

PLA2 acted preferentially on phosphatidylcholine, whilst non-toxic

PLAg showed no specificity (Napias and Heilbronn, 1980). Bee venom

PLA2 is centrally neurotoxic and exhibits behaviour intermediate

between non-toxic and neurotoxic phospholipases. The specificity of

neurotoxic PLA2 for brain preparations, as mentioned above, may reside

in the hydrophobic part of the molecule (the toxin's subunit B); it has

been reported that cholesterol may play some part in this specificity

(Napias and Heilbronn, 1980) and that the cholesterol to phospholipid

ratio may be important (Strong and Kelly, 1977). Strong and Kelly

(1977) also report that PLA2 activity is dependent on the physical

state of the phospholipid in the membrane and that 8-BuTx preferentially

hydrolyses membranes undergoing phase transition or that contain solid-

- fluid phase boundaries. The significance of B-BuTx's preference for membranes undergoing phase transition is not at present clear. However,

it has been noted that other hydrophobic proteins le.g. casein) show

preferential insertion into monolayers at their transition temperature

(Phillips et a K , 1975); it has also been suggested that hydrophobic

regions of the membrane may be partially exposed during such transitions

(Van der Bosch and McConnel, 1975) thus facilitating interaction of phospholipases with fatty acyl chains.

Recently, ^I-lab el le d (Rehm and Betz, 1982) and [3H] propionylated (Othman et al_., 1982) derivatives of 8-BuTx have been made, allowing partial characterisation of a specific membrane receptor for this toxin. Rehm and Betz (1982) report that chick brain synaptic 125 membranes contain a single class of saturable sites for I-8-3uTx with a Kq of about Q.5nM and density of 50fmol/mg of.protein. In the same year Othman et al_., (1982), using rat cerebrocortical synaptosomes, defined a saturable class of sites with Kg *t0.6nM and Bmax %150 fmol/mg of protein. Both of these studies suggest the involvement of a proteinaceous component in the observed specific binding due to its heat and protease sensitivities. Recently, 8-BuTx has been cross-linked to its specific binding protein in chick brain membranes (Rehm and Betz,

1983); it was shown to have an M of 95000. The major discrepancy between the above binding studies is that Rehm and Betz (1982) described a Ca^+-dependent interaction with brain membranes whilst Othman et al. (1982) found no such requirement for the divalent cation as measured biochemically or electrophysiologically; residual phospholi pase activity in the former preparation or species differences may account for this difference.

1.6.2 Venom toxins without PLAq activity. The physiological significance of the synaptosomal binding component(s) reported by Othman et al. (1982) was substantiated by the ability of toxin I, a protease inhibitor homologue from Dendroaspis polylepis polylepis (the black mamba) (Strydom, 1973), to completely inhibit the binding of

[ H]-8-BuTx to brain synaptosomes (X. *\.0.57nM). Toxin I (Mr of

7000) is devoid of PLA2 activity (unlike the other snake toxins mentioned above) and is an effective antagonist of the neuroparalytic action of 8-BuTx (Harvey, 1982); it has been shown to cause facilitation of ACh release from chick nerve-muscle preparations (Harvey and Karlsson, 1980). This protein is homologous to the B-subunit of 8-

BuTx and substantiates earlier proposals (Kondo et al_., 1978; Lee,

1979) that this subunit may be involved in specific binding to neuronal membranes. The inability of Rehm and Betz (1982) to illustrate 125 inhibition of 1-8- BuTx binding by isolated B-subunit may be due to conformational changes in the molecule that must occur on cleavage of the six or more disulphide bonds required for its separation from the

A-subunit. Another single polypeptide neurotoxin that is devoid of PLAg activity and chemically similar to toxin I, is dendrotoxin (DTx) from

Oendroaspis angusticeps (the Eastern green mamba); it consists of 59 amino-acid residues and is cross-linked by three disulphide bonds

(Harvey and Karlsson, 1980). A significant point of interest is the striking homology between the amino-acid sequences of toxins I and K

(from D. polylepis), DTx and the sequences of several protease inhibitors (Harvey and Karlsson, 1982). The aforementioned mamba toxins

(all of which are facilitatory on transmitter release) affect the physiological responses of nerve terminals to 8-BuTx, crotoxin and notexin but not taipoxin (Harvey and Karlsson, 1982); this infers that a separate binding domain is involved in the action of taipoxin.

DTx has recently been radio-iodinated and used in the characterisation of its specific binding sites in rat brain preparations

(Dolly et al_., 1984b). It is found to have a dissociation constant

(Kd ) of 0.3nM and Bmax %1200 fmol/mg of protein. In this study, the inhibition of I-labelled DTx binding by S-BuTx was pronounced although the interaction was of low affinity [K.^5 x 10’^M) This tends to suggest that B-BuTx and DTx do not bind to equivalent sites on synaptosomal membranes but may affect each other's binding by steric interactions. The DTx binding component on synaptosomal membranes is thought to be associated with the potassium channel; recent electrophysiological studies on hippocampal slices indicate that the pharmaco logical effects of DTx may be attributable to its ability to attenuate an outward K+ (A) current (Dolly et al., 1984b).

1.7 THE PRESENT STUDY.

This study concerns the use of botulinum neurotoxins (types A and

B) as probes for elucidating the underlying mechanism(s) of neuro transmiter release. A prerequisite for the use of these toxins in bio

chemical studies is their purification from native complexes with

haemagglutinating proteins (this is covered in Chapter 2); also in this

chapter, subunits from type A botulinum neurotoxin are separated and

reconstituted into an active form using a previously reported procedure.

A comparison is made between the structures and compositions of subunits obtained from types A and B neurotoxins (Chapter 3). Neurotoxin sub

units for amino-acid analysis were obtained by preparative SDS-PAGE.

Structural relationships between the various polypeptides were

investigated by peptide mapping; this was performed either by limited proteolysis in SDS-acrylamide gels or by reverse- phase HPLC of an enzymic digest.

Owing to the unsuitable nature of the neuromuscular junction for detailed biochemical studies, rat cerebrocortical synaptosomes were used as a model for the interaction of botulinum neurotoxins with neuronal tissues (Chapters 4 and 5). To study such interactions, types A and B neurotoxins were radio!abelled to high specific radioactivity with considerable retention of their biological activities and their satur able binding to presynaptic synaptosomal membranes was demonstrated

(Chapter 4). In Chapter 5, the nature and selectivity of this specific synaptosomal binding was investigated. The structure-function relation ships of these toxins, together with the characteristics of specific neuronal acceptors) in the CNS and at the NMJ are discussed (Chapter 6) in relation to the possible action(s) of these potent neurotoxins on transmitter release. - 46 -

CHAPTER 2

PURIFICATION OF NEUROTOXIN FROM CLOSTRIDIUM BOTULINUM TYPES A AND B *

♦

% 2.1 INTRODUCTION

A prerequisite for realising the enormous potential of botulinum neurotoxins as probes for studying the mechanism(s) of neurotransmitter release is their purification from complexes with non-toxic proteins in large quantities that would be necessary for such neurochemical work.

Cl. botulinum strains A, B and E are the most widespread, accounting for the majority of all recorded forms of human botulism (i.e. food, wound and infant) (Hatheway and McCroskey, 1981; Feldman, 1981; Sugiyama,

1980). Types A and B neurotoxins (like most of the botulinum toxins) exist in their native state as complexes with non-toxic proteins, one or more of which exhibits haemagglutinating activity (Kozaki et al_., 1974;

Sugiyama, 1980; Simpson, 1981a). These haemagglutinin components are found to protect the neurotoxic moiety from denaturation on ingestion

(Ohishi et al., 1977).

Much of the early work on the purification of botulinum toxin was carried out with Cl. botulinum type A. A crystalline form of the toxin was prepared which was considered to be a homogeneous protein with a molecular weight of 900000 (Lamanna et 31^ 1946). However, even at these early stages it was known to have two different biological activities, namely neurotoxicity and haemagglutinating activity

(Lamanna, 1959). In addition, it was shown that the haemagglutinin could be dissociated from the toxin without loss of toxicity and with an increase in specific activity (Wagman, 1954). Later, it was shown that these two activities could be separated by ion-exchange chromatography on DEAE-Sephadex A50 (DasGupta et al^., 1966) or DEAE-Cellulose (DasGupta and Boroff, 1967, 1968). The two resultant components, named a (toxin) and $ (haemagglutinin), were shown to have different molecular weights, lethalities and immunological properties. This was followed by a report that the 3-component could be resolved into three different aggregated forms with Mr of 290000, 500000 and 900000, as determined by gel filtration (DasGupta and Boroff, 1968).

The next major advance was the use of a crude extract, prepared by ammonium sulphate precipitation of culture filtrates, as the starting material for toxin purification (DasGupta et al_., 1970) instead of crystalline toxin. Neurotoxin was then prepared from this extract by a combination of ion-exchange chromatography and gel filtration.

Subsequently, a convenient method of purifying BoNT was reported

(Moberg and Sugiyama, 1978) which consisted of affinity chromatography on p-aminophenyl-B-D-thiogalactopyranoside immobilised on CH-Sepharose

4B. Galactose is reported to be one of the most potent inhibitors of haemagglutinin (DasGupta and Sugiyama, 1977b); thus the above analogue binds haemagglutinin very strongly. This purification procedure, using crystalline toxin complex, resulted in an essentially pure preparation of neurotoxin, although slight impurities were observed. In this laboratory* when either DEAE-cellulose (DasGupta and Boroff, 1968) or affinity (Moberg and Sugiyama, 1978) chromatography procedures alone were used to purify type A BoNT from its complexes with haemagglutinin, the neurotoxin contained variable amounts of a contaminating protein,

Mr %130000 (Tseet al_., 1982). Recently, a large scale procedure for the preparation of homogeneous BoNT (A) from a crude culture precipitate has been achieved by a combination of affinity and ion-exchange (using

DEAE-Sephacel) chromatography (Tse et al_., 1982). This procedure is now routinely used for the purification of type A neurotoxin.

The neurotoxic component (Mf % 160000) from type B botulinum toxin complexes has also been purified previously by gel filtration and ion-exchange chromatography (Kozaki and Sakaguchi, 1975; DasGupta and

Sugiyama, 1976) and was shown to comprise a mixture of single and dichain molecules. These preparations exhibit only part of their potential toxicity but may be fully activated by treatment with t^psin (DasGupta and Sugiyama, 1976); this also results in all single chain molecules being nicked to give two smaller proteins corresponding to the toxin's subunits (Mf of 51000 and 104000). In addition to outlining

the routine purification of type A BoNT, this chapter contains a

description of a relatively rapid method for the preparation of pure type B BoNT in high yield; it includes affinity and ion-exchange chromatography of crude culture acid precipitate, using a modification of that employed for BoNT (A).

No detailed study on the function of polypeptides from BoNT has yet been reported, although there is some indication that type B BoNT may bind to rat brain membranes through its larger subunit (Kozaki,

1979). Various microbial (e.g. diphtheria, cholera and tetanus) and plant (e.g. abrin and ricin) toxins contain heterologous subunits; in some of these, one subunit has been shown to be involved in specific binding to target tissues whilst the other type(s) of polypeptide is directly responsible for toxicity (Collier, 1975; Olsnes andPihl,

1982; van Heyningen, 1977; Wellhoner, 1982). To enable studies on structure-toxicity relationships and the assignation of function(s) to individual toxin subunits, the latter must be purified from the native toxin and renatured into an active form. Using a previously reported protocol (Kozaki et al^., 1981) of QAE-Sephadex ion-exchange chromato graphy, subunits from type A neurotoxin were separated and renatured.

2.2 METHODS

2.2.1 General Safety. Purification of neurotoxins from acid precipitates of cultures was carried out in a category B safety laboratory under negative pressure within the Vaccine Research and

Production Unit, Centre for Applied Microbiology and Research, Porton Down, Wiltshire. Protective gowns and gloves were worn at all times.

All procedures involving the handling of.large amounts of toxin within this laboratry were done inside a safety cabinet by the use of glove pockets (Melling and Allner, 1981). Toxic waste was either decontaminated with chloros (concentrated hypochlorite solution) or formaldehyde, whilst items to be recycled were autoclaved. All apparatus required for toxin purification and characterisation were permanently located within this laboratory. Decontamination of the whole laboratory was periodically performed by formaldehyde- vapourisation.

2.2.2 Purification of neurotoxin from Cl. botulinum type B by affinity and ion-exchange chromatography. Cl. botulinum type B (Okra) was grown for 48 hr in 201 cultures, after which toxin was precipitated by the addition of 3M to give a final pH of 3.0 - 3.5, and collected by continuous flow centrifugation as described earlier (Hambleton et al., 1981). Acid precipitated type B crude toxin (-ulOOg) was resuspended in 0.2M sodium phosphate buffer, pH 6.0 (300ml) using a

Colworth Stomacher homogeniser and extracted for lhr at room temperature, maintaining the pH at 6.0 with 1M NaOH. After centrifugation (30000 x g, 30 min at 4°C) the sediment was re-extracted under the same conditions and the toxin-rich supernatants pooled. This extract was treated with ribonuclease (100ug/ml, 1 hr at 34°C) prior to ammonium sulphate precipitation (the 20-60% sulphate cut was taken).

The precipitate was collected by centrifugation and resuspended in 60ml of 0.1M sodium phosphate buffer, pH 5.8, and dialysed against the same buffer overnight at 4°C. The dialysed toxin was centrifuged (30000 x g,

30 min at 4°C) and applied to a DEAE-Sephacel column (10 x 2.5cm), pre equilibrated with the latter buffer. During elution with 30ml of the same buffer, it was passed directly onto the affinity column (7 x 1cm) of p-aminophenyl-8-D-thiogalactopyranoside covalently coupled to CH-

Sepharose 4B (Moberg and Sugiyama, 1978). The extent of coupling

represented 8-11 urnol ligand/ml gel. After transfer of the toxin to the

affinity column, the DEAE-Sephacel column was disconnected and unbound

protein was washed from the affinity column with 60ml of 0 .1M sodium phosphate buffer, pH 5.8; the toxin was eluted subsequently with 0.15M

Tris-HCl buffer, pH 7.9, containing 1M NaCl. Fractions (2ml) with

A280nm > 0,5 were Pooled and dialysed overnight at 4 C against 0 .1M

Tris-HCl buffer, pH 7.9 (2 x 2 litres). The affinity-purified material was applied to a DEAE-Sephacel column (10 x 0.6cm) equilibrated in the same buffer, washed (with 20ml of this buffer) and eluted in two steps with 30ml of 0.1M Tris-HCl buffer, pH 7.9, containing 18mM NaCl followed by the same buffer containing 0.5M NaCl.

2.2.3 Isolation of type A neurotoxin by affinity and ion-exchange chromatography. Haemagglutinin-neurotoxin complexes were obtained from culture media acid precipitates by extraction, ammonium sulphate precipitation and DEAE-Sephacel ion-exchange chromatography as described by Tse et al_. (1982); except that the preabsorption of brown pigment present in the crude extract was carried out using swollen DEAE-

Sephacel (10% w/v, 10 min at 25°C) instead of DEAE-Sephadex A50 (50% w/v, several hours at 25°C). Purification of neurotoxin from these complexes with haemagglutinin by affinity and ion-exchange chromato graphy was carried out as reported earlier (Tse et a K , 1982). Affinity gel (p-aminophenyl-8-D-thiogalactopyranoside coupled to CH- Sepharose

4B) was equilibrated with 50mM sodium phosphate buffer, pH 6.3 and mixed batchwise with toxin, preequilibrated in the same buffer, for 2hr at room temperature with gentle stirring. The slurry was then packed into a column (10 x 0.6cm) and the buffer recycled once before its disposal.

After extensive washing with the same buffer O 5 0 m l ) the neurotoxin was eluted using lOOmM sodium phosphate buffer, pH 7.9, containing 1M sodium chloride. Affinity purified toxin, dialysed against 150mM Tris-HCl buffer, pH 7.9, was applied to a DEAE-Sephacel column (10 x 0.6cm) equilibrated in the same buffer. On washing with the aforementioned buffer, homogeneous neurotoxin was collected in the column void volume, whilst contaminating proteins remained bound. These contaminants may be eluted with 150mM Tris-HCl buffer , pH 7.9, containing 0.5M NaCI.

2.2.4 Native and SDS gradient-pore gel electrophoresis. Polyacrylamide slab gels (4-30%; PAA, 4/30 Pharmacia) were electrophoresed at 75Y for lhr prior to sample application. Native gel electrophoresis was carried out at 150Y (for 18h at 4°C), whilst electrophoresis in the presence of

SDS was performed at 125V (at 4°C) until the tracking dye had migrated off the gel, after which the voltage was lowered to 100V for 2hr. Under native conditions electrophoresis was performed in 90mM Tris-80mM boric acid, pH 8.2, containing 3mM EDTA. For electrophoresis in SDS, samples were heated (10 min at 60°C) in lOmM Tris-HCl, pH 8.0/lmM EDTA/2%

SDS/10% glycerol in the presence (reducing conditions) or absence (non reducing conditions) of 2-mercaptoethanol (5% v/v). The electrode buffer was 40mM Tris-20mM sodium acetate, pH 7.4/2mM EDTA/0.2% SDS.

Following electrophoresis, gels were fixed in 25% isopropanol/10% acetic acid, washed with deionised water and stained (^4h) with either

Coomassie blue R-250 (0.1% in 25% methanol/10% acetic acid) or Coomassie blue G-250 (0.04% in 3.5% perchloric acid); destaining was carried out with 10% methanol/10% acetic acid or 10% acetic acid, respectively.

Molecular weights of protein samples were estimated by comparison of their mobilities with those of protein standards (thyroglobulin, 669000 and 330000; ferritin, 440000, 220000 or 18500; catalase, 232000 or

60000; lactate dehydrogenase, 140000 or 36000; albumin, 67000; phosphorylase b, 94000; ovalbumin, 43000; carbonic anhydrase, 30000; trypsin inhibitor, 20100; oc-lactalbumin, 14400) as detailed in figure legends. 2.2.5 Two-dimensional gel electrophoresis. In the first dimension,

samples were subjected to disc gel isoelectric-focussing (IEF) according

to O'Farrell (1975). Gels were prepared using 1ml of 30% acrylamide

(28.4% acrylamide, 1.6% bis-acrylamide) solution, 40% Pharmalyte, pH 5-8

(0.4ml), 20% sucrose (4ml), TEMED (6pl) and deionised water (8ml). This

solution was degassed before the addition of 1.5% ammonium persulphate

(0.4ml); this solution was poured into glass tubes (11 x 0.3cm) and

allowed to set. The sample, in 2% Pharmalyte (pH 5-8) and 10% glycerol, was applied to the gel and overlayed with 1% Pharmalyte pH 5- 8/3%

glycerol (50pl). The electrode buffers were 20mM sodium hydroxide (at the cathode) and lOmM orthophosphoric acid (at the anode); the former buffer was degassed extensively before use. Samples were electrophoresed into the gels at 100V and run at 450V overnight (-ul2hr at 4°C).

Gels were then extruded from the tubes, cut longitudinally and one half sliced (2mm); each section was extracted with 0.5ml of lOmM KC1

(degassed) and the pH gradient determined. The other half of the focussed gel was equilibrated with SDS reducing sample buffer (5mM Tris

HC1, pH 6.5/2% SDS/5% 2-mercaptoethanol/10% glycerol) for 30 min at 4QC.

The equilibrated gel was placed onto a stacking gel on top of a 10% polyacrylamide gel and sealed in place with agarose (1% in the above sample buffer). SDS- polyacrylamide gel electrophoresis (SDS-PAGE) was performed using the discontinuous system of Laemmli (1970).

Stock solutions for the gels were prepared as below:

Solution A : 0.25M Tris-HCl buffer, pH 6.8/0.2% SDS

Solution B : 0.75M Tris-HCl buffer, pH 8.8/0.2% SDS

Solution C : 40% acrylamide (39% acrylamide, 1% bis-acrylamide)

Solution D : 1.5% ammonium persulphate.

A 10% acrylamide resolving gel was prepared with solutions B

(10ml), C (5ml), D (1ml), deionised water (4ml) and TEMED (15ul). After the separating gel had set the stacking gel was poured on top, consisting of solutions A (10ml), C (1.5ml), D (1.0ml), deionised water

(7.5ml) and TEMED (15jil). Electrophoresis buffer was 25mM Tris, 190mM glycine containing 0.1% SDS; gels were run at 120V (for -v8hr) until the ion front reached the end of the gel, then fixed and stained with 0.05%

Coomassie blue R-250 in 50% methanol/12% acetic acid. Gels were destained with 25% methanol/10% acetic acid followed by 10% methanol/10% acetic acid.

2.2.6 Ouchterlony double immunodiffusion. Gels (5 x 5 x 0.2cm) were prepared using 1% agarose in lOOmM sodium phosphate buffer, pH 7.0.

Centre wells contained horse antibodies (5pl) raised against botulinum neurotoxin-haemagglutinin complex type A or B. Protein samples (5ul) were placed in outer wells and left to diffuse for 24hr at 4'C. The gels were washed, fixed (10% acetic acid) and dried onto a glass plate.

Dried gels were stained for 10 min with 0.25% Coomassie blue R-250 in

40% methanol/10% acetic acid and destained with 10% methanol/10% acetic acid.

2.2.7 QAE-Sephadex chromatography of type A BoNT. Both subunits from

BoNT type A were separated by anion-exchange chromatography on diethyl-

(2-hydroxypropyl) aminoethyl (QAE)-Sephadex A50 in the presence of dithiothreitol (DTT) and urea (Kozaki et al_., 1981). Neurotoxin (3-5mg) was dialysed against sodium borate-phosphate buffer, pH 8.5 (28.8mM

Na2B^0y.IOH2O, 41.7mM NaH2P0^.2H20) and applied to a column (5 x 0.9cm) of QAE-Sephadex A50 equilibrated in the same buffer.

The column was washed with 20ml of buffer and then with 10ml of buffer containing lOmM DTT; this was followed by overnight treatment with 3ml of buffer containing lOOmM DTT and 2M urea. The column was washed subsequently with buffer containing lOmM DTT and 2M urea (%40ml) and then with the latter buffer containing 0.2M NaCl. Subunits were - 55 -

renatured by dialysis without agitation against 25mM sodium phosphate

buffer, pH 7.5, containing 0.1M NaCl for 43hr. Buffer solutions and gel

slurry were degassed prior to use and all procedures were carried out at

4°C.

♦ 2.2.8 Other determinations. Haemagglutinating activity was determined

in 50jil serially diluted samples using an equal volume of a 0.5%

suspension of chick red blood cells as described previously (Lowenthal * and Lamanna, 1956). Neurotoxic samples, serially diluted with 70mM

sodium phosphate buffer, pH 6.5, containing 0.2% gelatin were injected

(0.5ml) intraperitoneally into groups of 4 mice (% 20g) and the amount

♦ which killed 50% of the mice after 4 days (mouse LD^q ) determined.

Protein concentrations were determined colorimetrically using the Folin

phenol reagent (Lowry £t a K , 1951) or a Coomassie blue G-250 protein

dye assay (Bradford, 1976), using bovine serum albumin as a standard. ♦

2.3 RESULTS

+ 2.3.1 Purification of type B BoNT.

2.3.1.1 Affinity chromatography of crude toxin-haemagglutinin

complexes. Acid precipitation of toxic cultures (2 x 201) of Cl.

A botulinum type B was carried out by the addition of 3M H2SO4

to a final pH of 3.0 - 3.5 and the precipitate collected by

continuous centrifugation. The sedimented ,,acid-paste*‘ (%100g)

was extracted in 0.2M sodium phosphate buffer, pH 6.0 and showed

high levels of toxicity (1.8 x 1010 mouse LD5Q; Table 2.1).

After ribonuclease treatment, the toxic extract was concentrated

by ammonium sulphate precipitation (20-60% cut), resuspended in

60ml of 0.1M sodium phosphate buffer, pH 5.8 and dialysed against

the same buffer. Recovery of toxicity at this stage was about 60% - 56 -

Table 2.1 Purification of Cl. botulinum type B neurotoxin.

Toxicity Total Overal1 Purification Volume Protei n mouse Total LD50 /mg Recovery Stage (ml) (mg) protei n %

Suspension of 390 N.D. 8.0xl07 3.1X1010 N.D. 100 aci d-preci pi tated toxi n

0.2M sodium phos 600 7200 3.0xl07 l.8xl01° 2.5x10® 58 phate buffer, pH 6, extract

Resuspension of 101 3687 l.OxlO8 1.01x10*° 2.7x10® 33 20-60% ammonium sulphate precipitate

Affinity chromato 20 158 3.0x10® 6.0x10° 3.8xl07 19 graphy eluate

DEAE-Sephacel chromatography:

First peak (18mM NaCI) 6 4 0 . 9 7.5x10® 4.5x10° 1.1x10® 14.5 (2x10®)*

Second peak 10.5 57.8 l.OxlO7 1.05x10® 1.8x10® 0.3 (0.5M NaCI)

The first peak from DEAE-Sephacel chromatography of affinity- purified toxin was fully active after trypsinisation (5ug/ml, 30 min at 22°C); this also resulted in all single chain polypeptides being nicked to their dichain form. *

« Fig.2.1 Affinity and ion-exchange chromatography of type B toxin- haemagglutinin complexes.

(A) Chromatography of type B neurotoxin-haemagglutinin complex on DEAE-

Sephacel and p-amino phenyl-B-D-thiogalactopyranoside-Sepharose 4B.

The sample (3-4g protein) was applied to the DEAE-Sephacel column

in 0.1M sodium phosphate buffer, pH 5.8, eluted directly onto the

affinity column after which the former column was disconnected (1 ).

At this point toxin-complexes, not retained by the preliminary

DEAE-Sephacel column, had eluted onto the affinity column; further

elution of the DEAE-Sephacel column only increased the amount of

contaminating proteins passed onto the affinity matrix with no

significant increase in the amount of toxin present. Unbound

material was washed from the affinity column with the above buffer;

the neurotoxic material was subsequently eluted (2 ) with 0.15M

Tris-HCl buffer, pH 7.9, containing 1M NaCl; 1.5ml fractions were

collected.

(B) DEAE-Sephacel chromatography of affinity-purified type B toxin.

Affinity-purified material Kl60mg) was applied to the ion-

exchange column in 20ml of 0.1M Tris-HCl buffer, pH 7.9 and washed

with the same buffer (1). The column was then eluted with 0.1M

Tris-HCl buffer, pH 7.9, containing 18mM NaCl (2) and then with the

same buffer containing 0.5M NaCl (3); 1.5ml fractions were

collected during elution of the protein peaks. 0 v + H I i ♦ Fig.

Cn 0 0 I - 59 -

Fig. 2.2 SDS-PAGE of BoNT (B) at different stages of purification.

* 1234567 8 9 10

Samples were subjected to SDS-gradient pore (4-30%)-PAGE under

non- reducing conditions except for tracks 6 and 9, which were run under

reducing conditions (in the presence of 5% 2-mercaptoethanol). The gel

was fixed, stained with Coomassie blue R-250 and destained (as in

Methods). Track 1, marker proteins (from top to bottom): thyroglobulin,

ferritin (half unit), albumin, catalase, lactate dehydrogenase,

ferritin; tracks 2 and 10, marker proteins: phosphorylase b, albumin,

* ovalbumin, carbonic anhydrase, trypsin inhibitor, a-1actalbumin; track

3, toxin-haemagglutinin complex; track 4, eluate from affinity chromato

graphy; tracks 5 and 6, first peak from DEAE-Sephacel chromatography of

affinity purified material, track 7, second peak off the DEAE-Sephacel

column; tracks 8 and 9, trypsinised (5ug/ml, 30 min at 22°C) samples of

BoNT (B). of that contained in the phosphate buffer extract above; it had a specific neurotoxicity of 2.7 x 10^ mouse LD^/mg protein

(Table 2.1). This material was found to contain brown pigments that interacted with the affinity gel and decreased the specific absorption of haemaggluninin-toxin complex (> 50 % of applied toxin is eluted in the washthrough). These pigments were partially removed by DEAE-Sephacel chromatography; this enabled essentially all of the toxin complex to bind to the affinity matrix. Under the conditions used, the toxin was not retained by the DEAE-

Sephacel column and was eluted directly onto the affinity column, which bound more than 98% of the applied toxin. After dis connection of the DEAE-Sephacel column (Fig. 2.1A (1)) and thorough washing, a large proportion of the neurotoxin (% 60%,

Table 2.1) was then eluted from the affinity column (Fig. 2.1A

(2)) and had a specific toxicity of 3.8 x 107 mouse LD5Q/mg protein (Table 2.1). SDS-PAGE of this partially purified neuro toxin showed it to contain a major component (Mr ^160000) together with some minor protein bands (Fig. 2.2). Affinity- purified toxin possessed no haemagglutinin activity and gave a single diffuse line in double immunodiffusion tests (Fig 2.3).

2.3.1.2 DEAE-Sephacel chromatography of affinity purified material. Pure BoNT (B) was separated from contaminants present in the affinity purified toxin by a further chromatography procedure using DEAE-Sephacel (Fig. 2.IB). The pure neurotoxin, recovered in the first peak eluted with 0.15M Tris-HCl buffer, pH

7.9, containing 18mM NaCl (Fig. 2.1B(2)), had a high specific o toxicity of 1.1 x 10° mouse LD5Q/mg protein (Table 2.1) and gave a single sharp precipitin line on double immunodiffusion

(Fig. 2.3). Native-PAGE of highly purified type B BoNT showed a single diffuse band (Fig. 2.4) at a high molecular weight _(M Fig, 2.3 Ouchterlony double immunodiffusion gels of proteins purified

from type B toxin complexes.

The centre wells of the agarose gels contained horse antiserum

against crude type B toxin complex. Protein samples were placed in the

outer wells and allowed to diffuse (20 hr at 4°C) before washing,

fixing, drying, staining (with Coomassie blue R-250) and destaining.

Well 1, crude extract of acid precipitated toxin; well 2, resuspension

of material obtained by 20-60% ammonium sulphate precipitation of crude

toxin complexes; wells 3 and 7, affinity-purified toxin; wells 4, 5 and 8, first peak from DEAE-Sephacel chromatography; well 6, second

peak from DEAE-Sephacel chromatography. Fig. 2.4 Native gel electrophoresis of type B BoNT at different stages

of purification.

This was performed in 4-30% acrylamide slab gels in 90mM Tris-

80mM boric acid, pH 8.2, containing 3mM EDTA at 150V (18 hr at 4'C); they were fixed and then stained with Coomassie blue R-250. Track 1, protein markers (from top to bottom): thyroglobulin, ferritin, catalase, lactate dehydrogenase and bovine serum albumin; track 2, toxin-haemagglutinin complex; track 3, affinity-purified toxin; tracks

4 and 5, first and second peaks respectively from DEAE-Sephacel chromatography of affinity-purified material. Fig. 2.5 Two-dimensional gel electrophoresis of type B BoNT.

Purified type B neurotoxin (42pg) was run in the first dimension using disc gel isoelectric focussing (pH 5-8). The toxin was loaded at

100V and then run overnight U 1 2 hr at 4°C) at 450V. The gel was then sliced longitudinally, one half being sliced (2mm) for the calibration of pH (using lOmM KC1), whilst the other half was equilibrated in SDS- sample buffer containing 2-mercaptoethanol (see Methods) for 30 min at

4°C. This gel was then sealed on top of a 10% poly-acrylamide SDS-gel with 1% agarose (in the above sample buffer). After submitting the gel to electrophoresis, proteins were visualised using Coomassie blueR-250 as detailed earlier. Protein standards were run simultaneously in a separate track near the edge of the gel in the second dimension (t): phospjorylase b, albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibi tor. - 64 -

^250000) probably due to self aggregation of the toxin. On SDS-

PAGE run under non- reducing conditions, BoNT (B) gave a single

band with Mr of 160000 (Fig. 2.2). Under reducing conditions

three bands were observed (Fig. 2.2); the major component had a

* molecular weight identical to the non-reduced material and the

other two components (corresponding to the toxin's subunits) had

molecular weights of 107000 and 51000. Treatment of the neuro

toxin with trypsin (5ug/ml, 30 min at 22°C) increased the specific * Q Q activity from 1 .1 x 10° to 2 x 10° mouse LD^/mg protein

(Table 2.1) and resulted in the complete conversion of the neuro

toxin into the dichain form, as observed on SDS-PAGE under

♦ reducing conditions (Fig 2.2). Two dimensional gel electro

phoresis showed that the unnicked and nicked forms of neurotoxin

had indistinguishable isoelectric points (pi %6.0) (Fig. 2.5).

From the crude acid precipitate (total toxicity of 3.1 x 1010 ♦ q mouse LDj-q ), 4.5 x 10 mouse LD^q were recovered as pure

neurotoxin, representing a yield of 14.5% (Table 2.1).

In contrast, the second peak eluted from the DEAE-Sephacel column, * with buffer containing 0.5M NaCl (Fig. 2.1 B (3)), had a low

toxicity (1.8 x 10® mouse LD5Q/mg protein; Table 2.1) and

SDS-PAGE revealed the presence of numerous protein bands (Fig.

♦ 2.2). On double immunodiffusion, the material appeared as two

distinct lines (Fig. 2.3), one of which showed identity with the

pure neurotoxin. It was not possible to recover this small amount

* of neurotoxin left bound to the DEAE-Sephacel column after elution

of the first peak without co-elution of other proteins.

2.3.2 Purification of type A BoNT. Following extraction of culture acid

precipitates with phosphate buffer, 60% ammonium sulphate precipitation

and resuspension, much of the brown pigment present in this crude - 65 -

Table 2.2 Purification of Cl. botulinum type A neurotoxin.

Toxicity

Total Overall Volume Protein Total Recovery (ml) (mg) LD5Q/inl LD5Q LD5Q/mg %

0 .2M sodium phos- 455 4550 8.0xl07 3.6xl010 7.9xl06 100 phate (pH 6) buffer extract

Resuspension of 39 858 4.6xl08 1.8xl010 2.1xl07 50 » 60% ammonium sulphate precipitate after DEAE-Sephacel chromatography

Affinity * chromatography:

Unbound material 54 225 5.0x10s 2.7x10s 1.2 10( 0.8 Eluted peak 25.7 234 5.8x10s 1.5 1010 6.4 10 42

DEAE-Sephacel * chromatography:

First peak 22.9 30.5 2.7x10s 6.2x10^ 2 .0x10* 17.2 Second peak 11.5 138 1 .8xl07 2 .1 x10s 1.5xl06 0.6

% - 66 -

Fig. 2.6 Affinity and DEAE-Sephacel chromatography of toxin complexes

from Clostridium botulinum type A.

(A) Affinity chromatography was carried out on p-aminophenyl-B-D-thio- + galactopyranoside coupled to Sepharose 4B. Toxin-haemagglutinin

complex, dialysed against 50mM sodium phosphate buffer, pH 6.3,

was mixed with the affinity gel (equilibrated in the same buffer)

in batch (2 hr at 22°C), after which the column was packed and the

buffer recycled once through the gel before its collection as un

bound material. After extensive washing with the above buffer,

the neurotoxic fraction was eluted with lQOmM sodium phosphate

buffer, pH 7.9, containing 1M sodium chloride (|).

(B) DEAE-Sephacel chromatography of affinity-purified material. After m dialysis against 150mM Tris-HCl buffer, pH 7.9, eluted material

from affinity chromatography was applied to a DEAE-Sephacel

column, likewise equilibrated. Under these conditions pure

neurotoxin is eluted in the void volume whilst contaminant

proteins are retained by the gel and may be later eluted using the

above buffer containing 0.5M NaCl (\|/).

* In both (A) and (B), 1.5ml fractions were collected.

4 i. 2.6Fig. Absorbance (280nm) - 67 - 67 - Fig. 2.7 Analysis of BoNT (A) at different stages of purification by

PAGE.

Gradient pore (4-30%) acrylamide gel electrophoresis of protein

samples were carried out in slab gels under native conditions (gel A) or

in the presence of SDS, under non-reducing (gel B) or reducing (gel C)

conditions. Gels were fixed, stained with Coomassie blue G-250 and

destained as detailed in the Methods. Gels A, B and C: track 1, marker

proteins (in order of descending size), thyroglobulin, ferritin,

catalase, lactate dehydrogenase and albumin; track 2 , toxin-

haemagglutinin complex; track 3, affinity-purified neurotoxin; track

4, first peak from DEAE-Sephacel chromatography of affinity purified material. Gels B and C: track 5, marker proteins, phosphorylase b,

albumin, ovalbumin, carbonic anhydrase, trypsin inhibitor and o- lactalbumin. - 69 -

Fig. 2.7

♦

m extract was removed by preadsorption onto DEAE-Sephacel (10% w/v) for 10 min at 25UC. After DEAE-Sephacel chromatography of this crude complex

(the toxic fraction is not retained by the column) and ammonium sulphate precipitation, 50% of the original toxicity was recovered.

When the toxin complex had reacted with the affinity matrix, the column packed and the buffer recycled, < 2% of the applied toxicity was recovered in the column washings (Table 2.2). In excess of 80% of the applied toxicity was present in the neurotoxic fraction eluted with lOOmM sodium phosphate buffer, pH 7.9, containing 1M NaCl (Table 2.2;

Fig. 2.6A ). The affinity-purified toxin had a specific neurotoxicity of about 6.4 x 10^ mouse LD^/mg protein (Table 2.2), consisted of a major protein component (Mp **.140000) on analysis by SDS-PAGE (Fig.

2.7B) and showed a diffuse line on double immunodiffusion (Fig. 2.8); native-PAGE showed a diffuse (Mp 400000-600000) and a sharp (Mp

**.130000) protein band as major components (Fig. 2.7A). This affinity purified material was applied to a DEAE-Sephacel column preequilibrated with 150M Tris-HCl buffer, pH 7.9 (Fig. 2.6B); under these conditions, homogeneous BoNT was collected in the column void volume whilst impurities present were eluted in a single step using buffer containing

0.5M NaCl (Fig. 2.6B). The neurotoxin (Mp of 138000), homogeneous on

SDS-PAGE (Fig. 2.7B) and giving a single, relatively sharp, precipitin line on Ouchterlony gels (Fig. 2.8) had a specific neurotoxicity of 2 x o 10 mouse LDgQ/mg protein (Table 2.2). Under reducing conditions,

SDS-PAGE showed the dissociation of the neurotoxin into its two subunits

(Mp of 91200 and 55000) with intensities of staining proportional to their molecular size (Fig. 2.7C). On native-PAGE (Fig. 2.7A), the neurotoxin appeared as a diffuse band as seen in the affinity-purified toxin, suggesting it forms aggregates under these conditions. Overall recovery of toxicity in the neurotoxin fraction was generally between

15-20% of the original crude extract (e.g. Table 2.2). The non-toxic, 71 -

Fig. 2.8 Ouchterlony double immunodiffusion of proteins obtained during

purification of BoNT (A) from toxin-haemagglutinin complexes.

4 The centre wells of the agarose gel contained horse-antiserum

against crude type A toxin-haemagglutinin complexes. Protein samples

were placed in the outer wells and allowed to diffuse (20hr at 4aC) * before washing, fixing, drying, staining (with Coomassie blue R-250) and

destaining. Well 1, unbound material from the affinity chromatography

column; well 2, eluate from affinity chromatography; well 3, second

peak from DEAE-Sephacel chromatography of affinity purified neurotoxin;

wells 4 and 6, first peak from DEAE-Sephacel chromatography of affinity

purified toxin (pure neurotoxin); well 5, no sample. non-haemagglutinin contaminant proteins in the salt-eluted peak varied in their amounts present between the different culture precipitates used. The numerous proteins observed in this second eluted peak gave two precipitin lines on double immunodiffusion gels, both having non-identity with the pure neurotoxin (Fig. 2.8). The specific neuro toxicity of this fraction was generally about 10® mouse LD^/mg protein (e.g. Table 2.2), the toxicity being afforded by neurotoxin that remained bound to the gel matrix on washing with 150mM Tris-HCl buffer, pH 7.9.

2.3.3 Separation of subunits from BoNT (A) by QAE-Sephadex chromatography. In an attempt to separate the subunits of type A BoNT and reconstitute them in an active form, BoNT bound to a column of QAE-

Sephadex A50, was treated overnight with lOOmM DTT in the presence of 2M urea. The smaller of the toxin's subunits (L-subunit) was subsequently eluted with borate-phosphate buffer, pH 8.0, containing lOmM DTT and 2M urea. (Fig. 2.9, L), whilst the heavier subunit (H-subunit) remained bound. However, in the buffer prior to elution of the L-subunit there was another peak of absorbance at 280nm (Fig. 2.9, X); this was primarily due to the elution at this point of the high DTT concentrations employed (lOOmM; DTT, in its oxidised states, absorbs strongly in this region of the spectrum), although trace amounts of protein were present as shown by SDS-PAGE (Fig. 2.10). The eluted

L-subunit fraction contained this polypeptide as its major constituent

(Mr of 55000) although significant contamination by the H-subunit and intact toxin was observed (Fig. 2.10). On renaturation by dialysis against 25mM sodium phosphate buffer, pH 7.5, containing 0.1M NaCl, this

L-subunit preparation was found to have a specific toxicity of about

2 x 10® mouse LD^/mg protein, equivalent to 1% of that of the native toxin (Table 2.3). However, this value may be more than two-fold - 73

Fig. 2.9 QAE-Sephadex chromatography of type A BoNT under reducing

conditions in the presence of urea.

Neurotoxin (v3mg) was dialysed against sodium borate/phosphate * buffer, pH 8.5, and applied to a QAE-Sephadex-A50 column. Once bound,

the toxin was treated with the above buffer containing lOmM

dithiothreitol (DTT) and then overnight at 4°C with buffer containing

* lOOmM DTT and 2M urea. The first protein peak (L) containing the L-

subunit was eluted with borate/phosphate buffer containing lOmM DTT and

2M urea (1). The H-subunit was eluted in a second peak (H) using the

latter buffer containing 0.2M NaCl (2). Both fractions were renatured *• by dialysis (48 hr at 4°C) against 25mM sodium phosphate buffer, pH 7.5

containing 0.1M NaCl. The peak in absorbance (marked X) contains

negligible protein and is due to elution of the high concentrations

* (lOOmM) of DTT employed in overnight treatment of the toxin on the

column.

4 Absorbance (280nm) 2.9 Fig. L X - 74 - 74 - - 75 -

Fig. 2.10. SDS-PAGE of subunits from BoNT (A) separated by QAE-

Sephadex chromatography.

• - t f

1 2 3

After renaturation by dialysis against 25mM sodium phosphate buffer, pH 7.5, containing 0.1M NaCl, samples were applied to a gradient pore (4-30%) acrylamide slab gel and electrophoresed under non-reducing conditions (except for protein markers and track 3, which were run in the presence of 2-mercaptoethanol) as described elsewhere. The gel was then fixed, stained (with Coomassie blue R-250) and destained (see

Methods). Tracks 1 and 7, protein markers (phosphorylase b, albumin, ovalbumin, carbonic anhydrase and trypsin inhibitor); tracks 2 and 3, type A BoNT; track 4, material from QAE-Sephadex chromatography eluted in peak X, prior to the L-subunit; tracks 5 and 6, eluted peaks L and H respectively from QAE-Sephadex chromatography (see Fig. 2.9). - 76 -

Table 2.3 Separation and reconstitution of subunits from type A BoNT.

TOXICITY Total Protein (ug) LDsn/ml LDso/mg

BoNT 3000 6.0X107 2.0x10'

QAE-Sephadex chromatography:

Peak L 400 2.0xl07 2.2x10'

Peak H 650 4.0x104 2.7x10

Reconstituted I and II 14 4.0x10s 2.0x10 (after dialysis and centrifugation)

* higher depending on the degree of heterogeneity of this fraction with respect to intact toxin. The larger subunit (A-subunit, of 91200) was eluted from the QAE-Sephadex column with buffer containing 0.2M NaCl

(Fig. 2.9) and was shown to be essentially pure by SDS-PAGE (Fig.

2.10). Following renaturation, this fraction had a low specific 5 toxicity of 2.7 x 10 mouse LD5Q/mg protein, equivalent to 0.1% of the native toxin's activity; this residual toxicity is probably due to trace contamination by the latter protein. The recovery of proteins eluted from the ion-exchange column was about 50-60% of that originally applied.

The two subunits were renatured together (in approximately an equimolar ratio) by dialysis (without agitation) against 25mM sodium phosphate buffer, pH 7.5, containing 0.1M NaCl for 48 hr at 4°C. The specific neurotoxicity of this renatured subunit mixture was 2 x 10^ mouse LD50/mg protein (Table 2.3); this was equivalent to about 10% of the neurotoxin’s original activity.

On double immunodiffusion gels the H- and L-subunits were found to have differing antigenicities but each showing identity with native BoNT

(Fig. 2.11). The H-subunit gave a single sharp precipitin line which coalesced with the more diffuse line given by type A BoNT (e.g. Fig.

2.11, wells 1 and 6). The L-subunit gave a double precipitation (Fig.

2.11, well 2), one of which was non-identical to that of H-subunit but coalesced with that of the neurotoxin; the other line coalesced with the precipitin line of the H-subunit and is most probably the result of heterogeneity in this fragment (see Discussion). When the subunits were renatured together they gave a single precipitin line (although faint) which had identity with that from the H- and L-subunits (Fig. 2.11, wells 15 and 16); thus providing evidence that this separation and renaturation procedure does not affect the immunoreactivity of the proteins. Fig. 2.11 Quchterlony double immunodiffusion of type A neurotoxin subunits prepared by QAE-Sephadex chromatography.

Centre wells of the agarose gels contained purified horse IgG raised against crude type A toxin-haemagg|utinin complex. Toxin samples were placed in outer wells and left to diffuse (20 hr at 4°C) before washing, fixing, drying, staining (with Coomassie blue R-250) and destaining. Wells 1, 4, 7, 10, 11 and 13, peak H from QAE-Sephadex chromatography; wells 2, 5, 9, 12 and 14, peak L from the QAE- Sephadex % column; wells 3, 6 and 8, native type A neurotoxin; wells 15 and 16, native toxin reconstituted from the individual subunits obtained in the above separation. 2.4 DISCUSSION

The affinity and ion-exchange chromatographic procedure developed for type A BoNT (Tse et al., 1982) has been shown to be readily applicable to the purification of type B BoNT. The type B toxin-haemagglutinin complexes bound to the affinity gel and the eluted neurotoxin was devoid of haemagglutinin. The minor number of protein contaminants in this eluate were then efficiently removed by DEAE-Sephacel chromatography.

This procedure allows the rapid purification of Cl. botulinum type B neurotoxin in good yield (14.5%); it affords more than a two-fold increase in yield over previous purification methods employed (DasGupta and Sugiyama, 19/6; Kozaki et al., 1977). Pure BoNT (B), of high 8 specific neurotoxicity (1 .1 x 10 mouse LD^/mg protein) is obtainable from crude acid- precipitated toxin in less than 72 hr.

Analysis of type B BoNT by isoelectric focussing showed it to have a pi of 6.0, similar to that found for BoNT (A), pi = 6.5 (Tse et al., 1982).

The presence of three bands on SDS-PAGE when this neurotoxin was electrophoresed under reducing conditions (Mf of 160000, 107000 and

51000) is consistent with previous reports of this toxin derived from the same Clostridial strain (DasGupta and Sugiyama, 1976; Kozaki and

Sakaguchi, 19/5), where it was shown to be a mixture of single and dichain proteins. The specific activity oftrypsin-treated neurotoxin o (2 x 10 mouse LD^/mg protein), which consists solely of dichain molecules, is comparable with previously reported values for type B neurotoxin prepared by different methods (OasGupta and Sugiyama, 1977c;.

Kozaki et al^., 1977).

The purification of type A BoNT was carried out routinely as described by Tse et al^ (1982). The only modification made was in the pre-adsorption of. crude toxin complex with DEAE-Sephacel prior to application of the toxin sample to the initial ion-exchange chromatography column. The amount of DEAE-Sephacel (used in preference over

DEAE-Sephadex A50) employed in the pre-adsorption was reduced from 50%

(w/v) to 10% (w/v) and the time from several hours to lOmin at 25°C; this was sufficient to remove much of the brown non-toxic pigment present in the extract without the risk of losing excessive amounts of toxin complex due to non-specific adsorption. Recoveries and the specific activities of the various neurotoxic fractions correlate well with those previously reported. However, the higher specific toxicity o found for the homogeneous neurotoxin fraction (2 x 10 mouse LD^/mg protein) compared with the earlier report (8.3 x 10^ mouse LD^/mg protein) by Tse et jH. (1982), does not reflect upon the purity of the toxin obtained but is probably due to partial denaturation of the toxin- haemagglutinin complexes prior to their purification. On SDS-PAGE, under non-reducing conditions, the neurotoxin obtained here gave a similar molecular weight (Mr 138000) as that determined earlier (Mr

140000) (Tse et al_., 1982). Comparable sizes were also obtained for the toxin subunits, determined by SDS-PAGE under reducing conditions; in this report the Mr of the subunits were 91200 and 55000, compared to those calculated by Tse et al_. (1982) of 99000 and 55000. The slight discrepancy in size of the larger subunit may be due to the use of different molecular weight markers; using more appropriate markers the

Mr of the H-subunit was found consistently to be nearer the lower of the two values (i.e. 91200).

Using the affinity and ion-exchange- chromatography procedures described above for the purification of types A and B BoNT , very similar chromatography profiles were obtained (compare Figs. 2.1 and

2.6). Moreover, the major contaminating protein (Mr *ul30000). in the affinity-purified material of type A toxin complexes as seen on native- and SDS-PAGE (Fig. 2.7) was also present in affinity purified type B toxin (Figs. 2.2 and 2.4). In both cases, this material was eluted in - 81 -

the second peak from DEAE-Sephacel chromatography of affinity-purified

toxin (Fig. 2.2.; Tse et a1_., 1982). It would be interesting to know

how and when these proteins (as well as the haemagglutinin components)

become so tightly associated with the neurotoxin and whether they are

* synthesised by the bacterium at the same time. If these neurotoxins are

encoded by phage or plasmid genomes (see General Introduction), their

associated non-toxic proteins may also be; this may ensure stability of

the neurotoxic component directly after its translation and subsequent ♦ release into the extracellular environment.

Another possibility for the occurrence of the non-toxic proteins

of Mr -1.130000 is that they may have originated from intact BoMT; their

* presence after affinity chromatography may indicate that they also

exist, in their native state, as complexes with protein(s) having an

affinity for galactose. However, there is much evidence against this

idea; these proteins are single chain molecules, they are antigenically * distinct fom BoNT and appear to have a yellow pigmentation (although

this may possibly be due to a minor contaminant present). A non-toxic

variant of BoMT has not yet been described, but Cl. botulinum is known * to not always follow the 'one strain - one toxin' rule (Simpson, 1981a);

it is possible, albeit unlikely, that the types A and B organisms may

produce a non-toxic variant of BoMT in addition to their neurotoxic

form.

As a preliminary to studying the structure and function(s) of the

subunits from type A BoMT attempts were made to isolate them in

homogeneous forms using a published procedure (Kozaki et al^., 1981).

However, this method was not entirely satisfactory; heterogeneity of

the L-subunit, due to contamination with the H-subunit and native toxin,

was quite significant and problems also arose from the insoluble nature

of the L-subunit, except in very dilute solutions 100ug/ml). On

standing, eluted L-subunit in lOmM DTT and 2M urea formed an insoluble precipitate; this made the estimation of protein concentration and

recovery difficult and therefore reconsititution studies subject to

error., The discrepancy in the QAE- Sephadex chromatography elution

profile (Fig. 2.9) and that reported by Kozaki et al_. (1981) is

explained by the observation that the latter report expresses the eluate

in terms of protein concentration rather than absorbance units, thus

ignoring any change in absorbance due to interference by DTT (which, in

its oxidised forms, absorbs signficantly at A2g0nin^

The larger subunit (Mr % 92000), eluted with buffer containing

0.2M NaCl, was essentially pure on SDS-PAGE and was soluble in aqueous,

non- denaturing conditions. This protein gave a single sharp precipitin

band on immunodiffusion gels, showing its retention of immunoreactivity.

The double precipitin line given by the L-subunit on the same gels confirms its heterogeneity with respect to native toxin and H-subunit.

This data corresponds with previous reports that the individual subunits have different antigenic sites (Krysinski and Sugiyama, 1980). Although

the H-subunit demonstrated some residual toxicity (2.7 x 10® mouse

LD50/mg protein) this is thought to be contributed by the presence of minute quantities of native toxin. It has been shown for some other microbial and plant toxins, with similar molecular structure to botulinum neurotoxins (Collier, 1975; van Heyningen, 1977; Olsnes and

Pi hi, 1977) that the larger subunit is responsible for toxin binding and the smaller subunit for its toxicity. If this were the case with the

H-subunit from BoNT (A), one might expect the great molar excess of larger subunit over neurotoxin (in this preparation of H-subunit) to compete for neurotoxin binding sites and thus antagonise any action of native BoNT present in trace amounts. This was not found to be so; the

H-subunit preparation was toxic whether injected intraperitoneally or intramuscularly. It is possible that the large subunit retains some

inherent neurotoxicity but it is more likely that its ineffectiveness in Table 2.4 Summary of the structural properties of neurotoxins from Cl. botulinum types A and B .

Neurotoxicity Relative Immunoreactivity Molecular (mouse LD50/mg molecular Subunit Species forms protein) weight size E l BoNT Subunits

912000 Cl. botulinum type A all dlchaln 2xl08 138000 6.51 No cross-reactivity Antlgenlcally molecules 55000 with BoNT (B) dissimilar

Cl. botulinum type B mixture of 1 .1 x 108 160000 107000 6.0 No cross-reactivity Antigenically single and (single chain form) with BoNT (A) dissimilar2 dlchaln 2 x 108 51000 molecules (dichain form)

1 value from Tse et a K (1982). 2 reported by Kozaki and Sakaguchi (1975). competition with native toxin is contributed by the excessive number of sites that are thought to be present for botulinum toxins at synapses

(see General Discussion and Williams et al_., 1983); in addition, conformational changes probably occur on its separation from the L-subunit and renaturation, resulting in a decreased affinity for its acceptor. In subsequent chapters, the gross structural similarities that exist between BoNT (A and B), as summarised in Table 2.4, are examined in more detail. In addition, attempts are made to define the functional activities of the toxin subunits and to relate them to the toxin's effect on neurotransmitter release. - 85 -

CHAPTER 3 m

STRUCTURAL CHARACTERISTICS OF BoNT TYPES A AND B

♦

♦ 3.1 INTRODUCTION

Botulinum neurotoxins, as discussed previously (see General

Introduction) are a group of proteins which have very similar molecular weights, subunit structures (DasGupta, 1981) and pharmacological actions

(Simpson, 1981a). In addition to being immunologically distinguishable, although there appears to be some cross-reactivity between types C, E and F neurotoxins (Oguma et al_., 1982; Yang and Sugiyama, 1975), these toxins also exhibit differential intra- and inter-species toxicities

(Simpson, 1981a). Differences in the detailed structural character istics of these toxins must account for dissimilarities seen in their pharmacological and immunological properties; on the other hand, it would not be surprising if these closely related neurotoxins exhibited some structural homologies. To date very little work has been performed on any aspect of BoNT structure-function relationships.

The importance of the integrity of disulphide bonds in the toxicity of BoNT has been known for many years (Sugiyama et al_., 1973).

Reduction of these bonds causes complete loss of toxicity; however, it is still not certain whether the reduction of intra- or inter-molecular bonds is responsible for the loss of toxicity observed. More recently, homology in the position of some half-cystine residues in the poly peptides of BoNT (A and B) have been reported (DasGupta, 1981).

Chemical modifications of BoNT (A and E) have suggested that arginine residues and amino-groups are essential for neurotoxicity and immunoreactivity. DasGupta and Sugiyama (1980) showed that treatment of types

A and E neurotoxins with 1, 2-cyclohexanedione (which specifically modifies arginine residues under the conditions used) resulted in de toxification; the immuno-reactivity of type A BoNT was also altered as a result of such treatment. Later, DasGupta and Rasmussen (1981) showed that blocking amino-groups (viz. e-NH2-lysine and the a-NH2-group) - 87 -

group) wih 2-methoxy-5-nitrotropone (using about a ten-fold excess of

reagent over protein) also caused complete or partial detoxification of

BoNT types E and A respectively. This reagent did not alter the

serological activity of BoNT (E) but caused almost total disappearance

* of such activity in BoNT (A). These data suggest that arginine residues

and amino-groups are critical for the toxicities of BoNT (A and E) but

may have different roles to play in the serological activities of the

two neurotoxins; however, the different serologial activities may be a ♦ reflection of the different antibodies used.

The aforementioned studies involved the use of native neurotoxin

and thus changes in toxicity or serological activities cannot be

% ascribed to modification of particular subunits. So far, the only

comparisons in subunit structure between the different neurotoxin types

has been from studies on their amino-acid compositions (Type C BoNT

* only) or antigenic determinants. The amino-acid compositions of types A and B neurotoxin preparations (only the intact toxin was studied) were

first investigated about fifteen years ago (Beers and Reich, 1969;

Boroff et al_., 1970). More recently, amino-acid compositions for intact ♦ BoNT (E and F) (DasGupta and Rasmussen, 1983 a,b) and the separated

subunits from BoNT (C) (Syuto and Kubo, 1981) have been reported. In

the latter study the antigenicities of the subunits were found to be

♦ different, as already shown for types A (see Chapter 2) and B (Kozaki

and Sakaguchi, 1975) BoNT. The subunits of BoNT (C^) are said to have

different amino-acid compositions, the heavier polypeptide (H-subunit)

♦ containing a high content of acidic and neutral amino-acids whilst the lighter-subunit (L-subunit) contained more basic residues (Syuto and

Kubo, 1981). In contrast to this report, tetanus toxin is reported to

have subunits of very similar amino-acid compositions (Taylor et al.,

1983). These authors employed a quantitative method, developed

by Cornish-Bowden (1983), that uses a compositional index, S A n (defined - 88 -

in Methods), to test for possible relatedness between proteins of

similar size but of unknown sequence. If S A n < 0.42N, where M = the

total number of residues in a molecule, there is a very strong

possibility that the proteins are related; if 0 . 4 2 N < S A n < 0.93N,

* there is a weak indication that the proteins are related or if S A n >

0.93N, there is no reason to believe that a relationship exists. In the

case of tetanus toxin, comparing half the number of residues in the H—

subunit with the number of residues in the L-subunit, S A n was ♦ calculated as 60, were N=437 and thus 0.42N=184 (Taylor et al_., 1983).

Amino-acid analysis of proteins, in addition to providing an

overall picture of composition, may also give some indication as to fr which residues may be cleaved in order to produce a manageable number of

peptide fragments on which further structural studies may be performed.

Cleavage of proteins can be achieved by chemical (Jauregui-Adell and

* Marti, 1975; Walliman et a K , 1977) or proteolytic (Cleveland et al. 1977) methods. Such peptides can subsequently be resolved on

polyacrylamide gels or by chromatographic procedures. This technique of

peptide mapping is used frequently nowadays to discriminate between

proteins with very similar amino-acid compositions, accentuating

differences in the primary sequence of proteins. Peptide maps of the

different subunits from any BoNT type has not yet been reported.

However, in the case of tetanus toxin peptide mapping has been performed

in its separated subunits by limited proteolysis in SDS-acrylamide gels

and. by SDS-PAGE of cyanogen-bromide cleavage products (Taylor et al.,

1983); under these conditions similarities were observed in the

mobilities of some peptides obtained suggesting that at least some

degree of homology exists between these toxin subunits.

In the following study, compositional data is presented for subunits

prepared from BoNT (A and B) by SDS-PAGE. The relative proportions of

the different amino-acids present are compared in an attempt to gain - 89 -

Fig. 3.1 Cross-sectional view of the apparatus used for electroelution

of proteins from acrylamide gels.

Equilibrated slices of acrylamide gel (containing protein) are * placed in the wider arm of the sample holder containing electrophoresis

buffer; this is in contact with the cathode via the buffer reservoir

(50mM Tris-HCl buffer, pH 8.0, 0.1% SDS). During electrophoresis (120Y

for 20 hr at 4°C), proteins are eluted from the gel slices and retained

by the dialysis membrane at the anode end of the sample holder. The

buffer reservoir is continually circulated during electrophoresis using

a peristaltic pump. Eluted proteins may easily be removed with a * pasteur pipette; the buffer containing protein was agitated using the

pipette before its removal to minimise losses of protein on the dialysis

membrane. *

* r « * * * ♦ *

H**1 OQ • Protein sample in cu acrylamide gel Cathode Anode Sample holder

I VO 0 1

[_ Dialysis Electrophoresis Screw-cap membrane tank Rubber Buffer seal reservoir - 91 -

insight into the properties exhibited by the individual subunits (e.g. lytic activity, solubility). Additional information as to the occurrence of sequence homology between these subunits is provided by

peptide mapping of polypeptides prepared from native and carboxyamidomethylated BoNT (A and B) (CAM-BoNT (A and B)).

3.2 METHODS

3.2.1 Preparation of subunits from BoNT (A and B) by SDS-PAGE. For the

following procedures all glassware used was acid-washed and clean protective gloves were worn at all times. BoNT (A) and nicked BoNT (B)

(%1.5mg) were subjected to SDS-PAGE under reducing conditions as

described in Chapter 2 (Methods 2.2.5). Following electrophoresis,

protein bands were visualised by soaking the gel in 4M sodium acetate

(Higgins and Dahmus, 1979) and dissected out with a razor blade.

Electroelution of polypeptides from SDS-acrylamide gels was performed

using the apparatus outlined in Fig. 3.1. Gel pieces (sliced, not macerated through a syringe) were equilibrated in 50mM Tris-HCl buffer, pH 8.0, containing 0.1% SDS and placed in the wider arm of the sample holder, containing the same buffer. This arm was immersed in the cathode side of the buffer reservoir whilst the narrower arm was connected to the anode side. Electrophoresis was performed in the above buffer (120V constant, 20 hr at 4°C) which was continually circulated between the anode and cathode compartments of the electrophoresis tank using a peristaltic pump. Eluted proteins were retained by the dialysis membrane at the anode arm of the sample holder and were easily collected

using a pasteur pipette. Dialysis membranes were prepared by boiling in

2% (w/v) NaH2C0g and then twice in deionised water. Aliquots of eluted polypeptides were taken for protein assay, toxicity tests,

analysis by SDS-PAGE and amino-acid analysis. 3.2.2 Amino-acid analysis. Samples in acid washed hydrolysis tubes were dried under vacuum over P205 and resuspended in 50pl SM HC1 containing 0.1% phenol and 1 nmol of nor-leucine (internal standard).

Tubes were evacuated, sealed and hydrolysed at 110°C for 20 hr or a series of time points (21, 45, 70 and 120 hr). Following hydrolysis, samples were dried under vacuum, resuspended in lOOpl of 0.2M sodium citrate buffer, pH 2.2 and their composition determined on a Beckman 121

MB automated amino-acid analyser. The amino-acid content was quantified using the Beckman 126 data system or by measurment of peak heights. The number of half-cystines present was determined as cysteic acid after performic oxidation (Hirs, 1967). 30% (v/v) H202 (50pl) was incubated with 98% formic acid (950ul) for 2 hr at 25°C. Protein samples were dissolved in 98% formic acid (80|il) followed by the addition of methanol (20pl). Performic acid (50pl of the above solution) was added to the acidic protein solution at -10°C and incubated at this temperature for 2 hr. The reaction was stopped by dilution with deionised water (2ml); after lyophilisation the samples were hydrolysed

(20 hr) as above.

Tryptophan residues were determined spectrophotometrically as described by Edelhoch (1967). The absorbance of protein samples in 6M guanidine hydrochloride (GuHCl, ultrapure grade) was measured at 280nm and 288nm; the ratio of tryptophan to tyrosine residues present in the sample is given by the following equation:

Trp 1280.A288 - 385.A280

Tyr 4815.A280 - 5690.A288 Equation 3.1

The number of tyrosine residues is determined from the acid hydrolysate and thus the tryptophan content easily calculated. - 93 -

The compositional index SAn, described by Cornish-Bowden (1983),

was calculated from the following equation:

S A n = 0.5 2(n. -n. )2 - 0.035(N -N )2 + 0.535 In -N I Equation 3.2 ix iy x y 1 x jr I

* where X and Y are two proteins with N and N residues respectively, x y of which n^x in X and n ^ in Y are of the ith type of amino-acid.

* 3.2.3 Peptide mapping by limited proteolysis in SDS-acrylamide gels.

This was performed on subunits from native- and carboxyamidomethylated

(CAM)-BoNT (A and B) separated by SDS-PAGE. In the latter case type A

BoNT or nicked type B BoNT (%2mg) were incubated in the presence of 8M * urea and 5mM DTT for 30 min at 25°C. Iodoacetamide (lOmM final

concentration) was then added and incubated with reduced toxin for a

further 30 min at 25°C in the dark. Native- and CAM-BoNT were dialysed

* against 5mM Tris-HCl buffer, pH 6.8 , containing 0.5% SDS before being

subjected to SDS-PAGE under reducing and non-reducing conditions,

respectively (see Methods 2.2.5). The separated subunits were

visualised by soaking the gel in 4M sodium acetate and dissected out.

After washing out excess salt with deionised water KlOmin), gel pieces

were equilibrated in sample buffer (0.125M Tris-HCl buffer, pH 6.8,

containing 0.1% SDS and ImM EDTA) and applied to the stacking gel of a

12% acrylamide slab gel. This latter gel was prepared using the

solutions B, C and D detailed earlier (Methods 2.2.5):

Solution B 10ml * Solution C 6ml

Solution D 1ml

lOmM EDTA 2ml

Deionised water 1ml

TEMED 15ul The stacking gel and electrophoresis buffer used were as described before (Methods 2.2.5) except that both contained ImM EDTA. The gel pieces were overlayed with the above sample buffer containing 20% glycerol and then with buffer containing 10% glycerol, a-chymotrypsin

(0.4ug/track) and bromophenol blue. Electrophoresis was performed at

120v; when the bromophenol blue dye reached the end of the stacking gel the power was switched off for 30 min, after which the power was returned and electrophoresis continued until the tracking dye reached the end of the gel. Protein bands were stained with either Coomassie blue R-250 (see Methods 2.2.5) followed by silver staining or by silver staining alone.

3.2.4 Silver staining of SDS-acrylamide gels. This was carried out directly after electrophoresis or in some cases, after first staining with Coomassie blue R-250. The former staining procedure was that of

Morrissey (1981). Gels were soaked in 50% methanol/10% acetic acid and then in 5% methanol/7% acetic acid (30 min each), followed by washing with deionised water (30 min total with 3 changes of water). After treatment with 10% glutaraldehyde (30 min) gels were extensively washed in deionised water (at least 2 hr with several changes of water) before incubation (30 min) with DTT (lmg/200ml). This solution was replaced

(without washing the gel) with 0.1% silver nitrate (30 min) before addition of the developing solution (1.38ml of 8% formaldehyde in 600ml of 3% NagCOg); after two brief rinses in developing solution the bands were visualised in 200ml of the latter with gentle agitation.

When staining reached the required intensity the reaction was stopped by the addition of solid citric acid (24g).

SDS-acrylamide gels that had previously been stained with

Coomassie blue R-250 could subsequently be silver stained (Wray et al.,

1981). Destained gels were soaked in 50% methanol (at least lhr) and then in solution C for 15 min with gentle stirring. Solution C, made immediately prior to use, was prepared by adding solution A (0.8g silver nitrate in 4ml deionised water) dropwise to solution B (21ml of

0.36% NaOH plus 1.4ml of 14.8M NH^OH) and then increasing the volume to 100ml. After staining in solution C, the gels were washed with de ionised water (30 min, 3 changes of water) and the colour developed by the addition of freshly prepared solution D (2.5ml of 1% citric acid added to 0.25ml of 38% formaldehyde and the volume increased to 500ml with water). The reaction was stopped by placing the gel in 10% methanol; gels were washed in deionised water and stored in 50% methanol.

3.2.5 Preparation of subunits from carboxyamidomethylated (CAM)-BoNT (A and B) by high pressure liquid chromatography (HPLC). CAM-BoNT (A and

B) were prepared as described above except that 50pCi iodo-[l-^C]- acetamide was added to the reduced toxin sample (30 min at 25°C) before the addition of excess unlabelled iodoacetamide. Following dialysis against 6M GuHCl and concentration with dry Sephadex, CAM-BoNT was

injected (lOOpl aliquots) onto a 60cm TSK G4000 SW sizing column (Yarian

Associates) preequilibrated in 6M GuHCl (degassed before use; ultrapure

grade, Bethesda Research Laboratories, Cambridge). Polypeptides were eluted at a flow rate of 0.8ml/min and the absorbance at 280nm recorded;

protein peaks from several injections were pooled before concentrating

by vacuum dialysis and subsequent dialysis against 0.5M acetic acid, pH

3.2. Aliquots of these CAM-subunit preparations were taken for toxicity

assay and analysis by SDS-PAGE.

3.2.6 Peptide mapping of CAM-subunits by reverse-phase HPLC. Poly

peptides obtained from CAM-BoNT (A and B) by HPLC (see above) were dried - 96 -

under vacuum and resuspended in 150pl 0.1M NH^HCO^ containing 2M

urea; the pH was adjusted to7.5 with 0.25M acetic acid. Enzymic

digestion of subunits was performed with Staphylococcus aureus V8

protease (20pg/sample) for 18 hr at 37°C. Protein digests were directly m applied to a Synchropak RPP C1Q reverse-phase HPLC column

(7.5x0.46cm); pore size 300A, particle size, 5pm; Synchrom, Linden,

Indiana) equilibrated in 0.1% trifluoroacetic acid. Peptides were

eluted in a linear gradient of acetonitrile (0-50%) over 1 hr at a flow

rate of 1.5ml/min. Fractions were collected at 0.5 min intervals and

the A2Qgnm recorded on a chart'recorder. More detailed information of

each peptide map was permanently recorded on a computer disc using a

Hewlett-Packard data system; plots of the different maps monitored at

various wavelengths could then be accurately superimposed at a later

date. Aliquots (20pl) from each fraction were added to 5ml of Ready—

* Solv HP/b (Beckman) and submitted for scintillation counting. Fractions

containing peptides of interest were lyophilised and the amino-acid

content determined by acid hydrolysis (20 hr) as described previously.

♦ 3.2.7 Protein determination in the presence of SDS. Proteins in the

presence of SDS were quantified colorimetrically using a modification by

Markwell et al^. (1978) of the Folin-phenol assay (Lowry et al^., 1951): ♦ Solution A: 2% Na2C03

0.4% NaOH

0.16% sodium tartrate

m 1% SDS

Solution B: 4% CuSO^.SHgO

Solution C: Add 250pl. solution B to 25ml solution A

Solution D: Folin and Ciocalteu's phenol reagent (1:1

diluted with deionised water). Solution C (600jil) was added to the sample (200pl) and incubatd for a.15 min at 25°C; solution D (60ul) was then added and the colour allowed to develop for 45 min at 25°C before reading at AygQnm. Bovine serum albumin in the presence of the appropriate concentration of SDS was used as a standard.

3.3 RESULTS

3.3.1 Isolation of subunits from types A and B BoNT by preparative

SDS-PAGE. In order to investigate the protein chemistry of botulinum

neurotoxins, their individual subunits must first be isolated in a pure

form. Isolation of both subunits from BoNT (A) by ion-exchange

chromatography was not totally satisfactory (see Chapter 2). Therefore,

preparative SDS-PAGE was employed to circumvent problems arising from

cross-contamination of the toxin’s polypeptides. Subunits from native

BoNT (A and B) were prepared in this manner under reducing conditions

and visualised readily by soaking the gel in 4M sodium acetate for about

10 min; high salt concentrations precipitate SDS not bound to protein, whilst protein-bound SDS remains clear. The polypeptides were electro- eluted fom gel slices using the apparatus described in Fig. 3.1. Total

recovery of protein was generally between 30-40% of that applied to the acrylamide gel, as determined by a modification of the Folin assay for

use with proteins in the presence of SDS; both subunits were recovered

in similar proportions. Eluted proteins were found to be concentrated

in the buffer above the membrane at the anode end of the sample holder

as the remainder of the buffer contained minimal amounts of protein

« 1 0 % of the total protein eluted). When assayed for toxicity these

subunit preparations (from types A and B neurotoxins) were non-toxic at

the lowest dilution tested (i.e. < 1 0 mouse LD5Q/mg protein);

however, any effect of SDS or the high salt concentrations employed on - 98 -

Fig. 3.2 Purity of subunits obtained from native BoNT, types A and B,

by preparative SDS-PAGE.

1 2 3 4 5 6 7

BoNT (A and B) were subjected to SDS-PAGE under reducing

conditions. Protein bands corresponding to the toxin's subunits were

visualised by soaking the gels in 4M sodium acetate. After excision of ♦ these bands, proteins were collected by electroelution in Tris-acetate

buffer, pH 8.0, containing 0.1% SDS, for 20 hr at 4°C. Aliquots (4-8ug

protein/track) were submitted to SDS-PAGE to ascertain their purity. * Proteins were visualised either by staining with Coomassie blue R-250

(tracks 1-5) or by silver staining (tracks 6 and 7). Tracks 1, 4 and 6,

protein standards (phosphorylase b, albumin, ovalbumin, carbonic

anhydrase, trypsin inhibitor); tracks 2 and 3, H- and L-subunits,

respectively, from type B BoNT; tracks 5 and 7, H- and L-subunits,

respectively from type A BoNT. -99-

Table 3.1 Amino-acid compositions of the subunits from type A BoNT.

Heavy Chain (M_ 91200) Light Chain (M_ 55000)

Residues Percent of Residues Percent of Amino acid per mol total residues per mol total residues

Half-cystine 6a 0.7 7a 1.5 Aspartic acid 16.4 13.1 132b 63b Threoni ne 45h 5.6 33“ 6.9 Seri ne 50b 6.2 27 b 5.6 Glutamic acid 83 10.3 46 9.6 Proline 23 2.9 21 4.4 Glycine 46 5.7 36 7.5 Alanine 37. 4.6 23 r 4.8 Valine 56 7.0 30c 6.2 Methionine 5 0.6 3 0.6 9.8 30c Isoleucine 79c 6.2 Leucine 71c 8.8 39c 8.1 Tyrosine 42 5.2 25 5.2 Phenylalanine 33 4.1 32 6.7 Lysine 56 7.0 36 7.5 Histidine 8 1.0 8 1.7 Arginine 3.5 3.1 28d 15d Tryptophan 3d 0.4 7d 1.5

Total residues 803 100.0 481 100.0

Polar amino acids Basic 92 11.5 59 12.3 Acidic and neutral 404 50.3 237 49.3

Hydrophobic amino- acids 307 38.2 185 38.5

The data was obtained from a single preparation of heavy and light chains from type A neurotoxin. Mean values are given for 2 or more determinations made after hydrolysis of samples for 21, 45, 71 and 120 hr, except where stated. a Determined as cysteic acid after oxidation with performic acid (20 hr hydrolysis) (Hirs, 1967). k Extrapolated to zero time (t=0). c Extrapolated to infinite time (i.e. 1/t 0).

^ Determined spectrophotometrically (Edelhoch, 1967). -100-

Table 3.2 Amino-acid compositions of the subunits from type B BoNT.

Heavy Chain (M 107000) Light Chain (M„ 55000) — r------

Residues. Percent of Resi dues Percent of Amino acid per mol total residues per mol total residues

Half-cystine 9a 1.0 ia 0.2 15.6 Aspartic acid 144 65h 14.6 Threonine 38b 4.1 16bh 3.6 Serine 65 b 7.0 23 b 5.2 Glutamic acid 101 10.9 46 10.3 Pro!i ne 21 2.3 19 4.3 Glycine 58 6.3 27 6.1 Alanine 34 3.7 14 3.1 Vali ne 40c 4.3 20c 4.5 Methionine 9 1.0 7 1.6 Isoleucine 110 c 11.9 63c 14.2 Leuci ne 79 c 8.5 39c 8.8 Tyrosine 56 6.1 22 4.9 Phenylalanine 49 5.3 25 5.6 Lysine 72 7.8 39 8.8 Histidine 9 1.0 4 0.9 Argi ni ne 27rt 2.9 14. 3.1 Tryptophan 4d 0.4 ld 0.2

Total residues 925 100.0 445 100.0

Polar amino acids Basic 108 11.7 57 12.8 Acidic and neutral 471 50.9 200 44.9

Hydrophobic amino- acids 346 37.4 188 42.2

The data was obtained from a single preparation of heavy and light

chains from type B neurotoxin. Mean values are given for 2 or more

determinations made after hydrolysis of samples for 21, 45, 71 and 120

hr, except where stated.

a Determined as cysteic acid after oxidation with performic acid (20 hr hydrolysis) (Hirs, 1967).

k Extrapolated to zero time (t=0).

c Extrapolated to infinite time (i.e. 1/t ^0).

^ Determined spectrophotometrically (Edelhoch, 1967). denaturation was not evaluated. The purity of these subunits was demonstrated by SDS-PAGE (Fig. 3.2). Only a single band was observed for the H-subunit from types A and B BoNT, but a very faint protein band of higher molecular weight was apparent in the L-subunit preparation from BoNT (B) (Fig. 3.2, track 3); 'heterogeneity' was more apparent in the preparation of L-subunit from BoMT (A); however, the higher molecular weight protein (Mr ^105000) is thought to be a dimer of the

L-subunit (Mf = 55000 x 2 = 110000), arising from reoxidation of cysteine residues to form intermolecular disulphide bonds. One must note that the sample was electrophoresed under non-reducing conditions; in hindsight this was probably a mistake. Contamination of this preparation by H-subunit is not thought likely owing to the greater mobility of the latter protein (compare Fig. 3.2, tracks 5 and 7 with protein markers in tracks 4 and 6).

3.3.2 Amino-acid compositions of subunits from native BoNT (A and B).

These were determined for the subunits of native BoNT (A and B) obtained by preparative SDS-PAGE (Tables 3.1 and 3.2). On comparing the percentage of individual amino-acids present in the H- and L-subunits from type A BoNT (Table 3.1) only a few distinct differences were observed, namely in proline, isoleucine and phenylalanine residues.

Similarities were also apparent between the H- and L-subunit of type B

BoNT (Table 3.2); but notable differences were apparent in the proportion of serine, proline and isoleucine present. Smaller differences in the number of tyrosine and lysine residues may also be significant.

A more general comparison was made of the different types of amino-acids present, classified according to their 1R* groups; the polypeptides of BoNT (A) seemed almost identical (Table 3.1) although the L-subunit of BoNT (B) had a slightly higher proportion of hydro phobic residues and slightly lower amounts of acidic and neutral residues when compared to the H-subunit from type B BoNT (Table 3.2).

In addition, the H-subunits of types A and B BoNT (A and B) showed greater similarities than the L-subunits of these toxin types. Owing to the very small amounts of protein available for analysis, and the greater number of procedural steps involved, the determinations of tryptophan and half-cystine residues were subject to greater errors than the other residues.

The compositional index SAn, described by Cornish-Bowden (1983), was used to determine any relatedness between the different polypeptides by their amino-acid compositions. To test for this between the H- and

L-subunits of the same neurotoxin, the number of each residue in the

L-subunit was compared to half the number of the same residue in the

H-subunit. In the case of subunits from type A BoNT, S A n = 381 and N =

481, whereas for type B BoNT, S A n = 219 and N = 445. Hence for both pairs of polypeptides 0.42N < S A n < 0.93N, giving "a weak indication that the proteins are related". When a comparison was made between the two H- subunits and the two L-subunits, S An = 309 < 0.42N and S An =

851 > 0.93N, respectively. Concerning the latter calculation of S a n, there may be an erroneous estimation of the number of isoleucine residues present in the L-subunit from BoNT (B); compare the number of isoleucine residues in the L-subunit from type A BoNT (Table 3.1) and type C BoNT (Syuto and Kubo, 1981). This apparent difference in iso leucine residues between the two L-subunits is the main contributory o factor towards such a large S A n value (i.e. (nT, - nT1 ) = 1089).

If S A n is recalculated, omitting values for isoleucine, then 0 . 4 2 N <

S A n = 241 < 0.93N; this would suggest that there may be some related ness between these two polypeptides. - 103 -

Fig. 3.3 Comparison between electrophoretic peptide maps of subunits

obtained from types A and B BoNT.

Subunits of BoNT (A and B) were separated by SDS-PAGE. After m visualising with 4M sodium acetate, the bands were cut out, washed and

equilibrated (30 min) in sample buffer (0.125M Tris-HCl buffer, pH 6.8 ,

0.1% SOS, lmM EDTA). Sample wells of the stacking gel were filled with

the above buffer before insertion of the gel pieces. Samples were over-

layed with buffer containing 20% glycerol and then with buffer

containing 10% glycerol and a-chymotrypsin (0.4ug/well); protein

standards were run in the absence of protease. Polypeptides were * resolved on a 12% acrylamide slab gel. Electrophoresis was carried out

as described previously, except that the current was switched off for 30

min when the bromophenol blue dye approached the end of the stacking

gel. After completion of electrophoresis the gel was stained with

Coomassie blue R-250, destained and then silver stained (Wray et al.,

1981). Track 1, standard proteins stained with Coomassie only (from top

to bottom: phosphorylase b, albumin, ovalbumin, carbonic anhydrase,

trypsin inhibitor and a-lactalbumin); tracks 2 and 3, H-subunit from

BoNT (A and B) respectively, tracks 4 and 5 , L-subunit from BoNT (A and

B) respectively; track 6, a-chymotrypsin alone. Arrows indicate * peptides from H- or L-subunits of similar size.

* - 104 -

Fig. 3.3

♦

3 5 m

4 - 105 -

Fig. 3.4 Similarities in the peptide maps of subunits from CAM-BoNT (A

and B).

These were obtained from CAM-BoNT (A and B) by limited proteolysis 4 in SDS-acrylamide gels as described in Fig. 3.3; following electro

phoresis gels were silver stained (Morrissey, 1981). Electrophoresis of

proteins in tracks 7-11 was performed in the presence of a-chymotrypsin

whilst proteins in tracks 1-6 and 12 were run in its absence. Tracks 1

and 8, H-subunit from type A CAM-BoNl; tracks 2 and 10, L-subunit from

type A CAM-BoNT; tracks 3, 6 and 12, protein standards (see Fig. 3.3);

tracks 4 and 9, H-subunit from type B CAM-BoNT; tracks 5 and 11, w L-subunit from type B CAM-BoMT; track 7, a- chymotrypsin only. Arrows

indicate peptides with similar electrophoretic mobilities.

* 106

Fig. 3.4 I • • « u o> * *

TT r T TT

04 1 I t • 1 9 1 1*4 « * * y * ♦ n m I o

Ol t I • 1 t 1 t«► " ----

1*4 • * y t t tt| j •4*##* 1 4 T TT T T ^

M i l l 11 1 \L 1

C 7 n

WR**4tf* * * i4 B OO ♦1

i 4 < m sO A ^ - - ♦ ♦ i - t V i_ n l^v (

m 1 # ♦ # ♦ 1 ^

: 4 -- o i 4 4 • > 3.3.3 Electrophoretic peptide mapping of constituent subunits of types A and B neurotoxins. One-dimensional peptide maps were obtained for partially proteolysed subunits from native- and CAM-BoNT (A and B).

Simultaneous mapping of protease digests from the H- and L-subunits of

BoNT (A and B) on the same acrylamide gel allowed an unambiguous comparison to be made. The peptide maps obtained after partial digestion of subunits from native BoNT (A and B) with a-chymotrypsin are shown in Fig. 3.3. The protein standards are illustrated after

Coomassie staining only, as subsequent silver staining produced excessively large bands owing to a greater amount of protein applied to this gel track relative to the others. Several peptides of similar size were observed in maps of both H-subunits (Fig. 3.3, tracks 2 and 3) or

L-subunits (Fig. 3.3, tracks 4 and 5). This tends to suggest at least

some structural similarities in each of the two different H- and L-

subunits. In addition, similarities were apparent between the H- and L-

subunits of type B BoNT (Fig. 3.3, tracks 3 and 6), although this was not as evident in the subunits of type A BoNT. The presence of minor

proteinaceous components with mobilities slower than the intact subunits

suggest some aggregation of polypeptides; this may result from

oxidation of cysteine residues to intermolecular cystines during the

peptide mapping procedure, which is performed in the absence of

reductant. To avoid interferences by such aggregation, peptide mapping was performed under similar conditions using subunits separated from

CAM-BoNT (A and B). As shown in Fig. 3.4 (tracks 1, 2, 4 and 5), the

subunits prepared from CAM-BoNT were pure with negligable or no protein

bands at higher molecular weights. The proteins present in minimal

quantities staining up at lower molecular weights are probably the

result of proteolysis of subunits by trace amounts of a-chymotrypsin

from neighbouring tracks; the polypeptide fragments have similar

mobilities to some of the bands seen in peptide maps of the respective subunits. On comparison of the H-subunits (Fig. 3.4, tracks 8 and 9)

and L-subunits (Fig. 3.4, tracks 10 and 11) from CAM-BoNT (A and B)

considerable similarities in the number of peptides of the same molecular weight were observed; in both instances these amounted to

approximately 25% of the number of peptides present. Moreover, the

similarities observed between peptide maps of subunits from native BoNT

(B), as seen in Fig. 3.3 (tracks 3 and b) were more clearly defined when

CAM-subunits were used (Fig. 3.4, tracks 9 and 11). Fewer, but still a

significant number (%25%) of the peptides resolved in the L-subunits

from type A CAM-BoNT (Fig.3.4, tracks 8 and 10). These may have been

present when subunits from native-BoNT were mapped but, owing to poorer

resolution of peptides in the former study, were not as apparent.

Collectively, these results suggest homologies in the primary structures

of H-subunits and L-subunits from BoNT of two different botulinal

strains; also, there is evidence to support the view that some homology

exists between the H- and L-subunits of the same type of BoNT.

3.3.4 Rapid preparation of polypeptides from CAM-BoNT by HPLC.

Purification of BoNT subunits by conventional permeation and ion-

exchange chromatography methods are generally unsatisfactory; poor

resolution of peaks results in cross-contamination of proteins, which

are eluted in large volumes and recoveries tend to be rather low due to

non-specific absorption to gel matrices. The aforementioned procedures

are also very time consuming. In an attempt to overcome these

difficulties, high pressure permeation chromatography was performed on

types A and B CAM-BoNT in the presence of 6M GuHCl. Alkylated and

hence, inactivated, forms of neurotoxin were used because access to HPLC

apparatus was possible only in an open laboratory; under such circum

stances the toxin must contain negligible activity « : 10^ mouse

LDg0/mg protein), which these preparations complied with, in order to - 109 -

Fig. 3.5 Rapid preparation of subunits from CAM-BoNT types A and B by

HPLC under denaturing conditions.

Toxin samples in 6M urea were reduced with dithiothreitol and

treated with iodo-[l- CJ acetamide. After dialysis against 6M GuHCl

(overnight at 4°C) and concentration, aliquots (lOOpl) were injected

onto a TSK G4000SW column (60cm) likewise equilibrated. The column was

run in 6M GuHCl at a flow rate of 0.8ml/min and the eluted polypeptides

collected manually; corresponding protein peaks from many runs were

pooled prior to concentration and dialysis. A) type A CAM-BoNT and B)

type B CAM- BoNT; 1, H-subunit and 2, L-subunit from respective

toxins.

♦

* U jUJ U lli) * * * • * * ♦

+ i. 3.5 Fig. Fig, 3.6 Purity of the subunits obtained by HPLC.

Polypeptides eluted from a TSK G4000SW HPLC column were concentrated by vacuum dialysis and dialysed against 0.5M acetic acid, pH 3.2. Aliquots of concentrated and dialysed samples were adjusted to pH 6.5 and diluted into SDS-sample buffer (non-reducing). SDS-PAGE was performed on a 10% acrylamide slab gel and proteins were stained with

Coomassie blue R-250 as described earlier. Tracks 1 and 6, protein standards (see Fig. 3.2); tracks 2 and 3, H- and L-subunits respectively from type A CAM-BoNT; tracks 4 and 5, H- and L-subunits from type B CAM-BoNT respectively. keep within safety limits concerning the handling of botulinum toxins in non-containment laboratories. In addition, complications may arise from reoxidation of half-cystine residues as already seen in peptide mapping studies (Fig. 3.3). A TSK G4000 SW column (60cm) was able to resolve the H- from the L-subunit of CAM-BoNT (A and B) although the separation was not to the optimal, baseline levels (Fig. 3.5). The small amount of residual intact toxin (Mr %150000), present in both CAM-BoNT samples, was eluted prior to the H-subunit either as a broad shoulder (Fig. 3.5A) or as a small partially resolved peak (Fig 3.5B); most of this intact toxin was collected separately from the H-subunits and discarded. The relative recoveries of H- and L-subunits from type A CAM-BoNT as indicated by their absorbance at 280nm were found to be similar (Fig.

3.5A) although with CAM-BoNT (B) a lower recovery of L- subunit was noticed (Fig. 3.5B). The latter observation may be owed to greater non-

-specific absorption of the L-subunit to membranes during dialysis and sample concentration prior to injection onto the column. The purity of subunits prepared by HPLC was assessed by SDS- PAGE (Fig. 3.6). All four subunits from CAM-BoNT (A and B) were relatively pure with only

slight cross-contaminations present. This heterogeneity could easily be improved upon by more selective pooling of eluted material and/or re chromatography. After concentration of the eluted polypeptides by vacuum dialysis and dialysis against acetic acid their protein contents were assayed; recoveries of proteins from HPLC were estimated at

80-90%. However, some protein was found to precipitate out in both the

H- and L-subunit preparations on dialysis against acetic acid; in view of this, such acidic conditions should be avoided in future studies. At

a later date, if samples are to be prepared by HPLC for use in the type

of peptide mapping studies described below, then dialysis ought to be

against the buffer used in enzymic digestions; i.e. i.e. 0.1M ammonium

bicarbonate buffer, pH 7.5, containing up to 4M urea (Staph, aureus V8 protease exhibits 50% of its original activity in this concentration of urea).

3.3.5 Comparison of reverse-phase HPLC peptide maps obtained from CAM- subunit digests. Additional information on similarities that exist between subunits of CAM-BoNT (A and B) was obtained from Staph, aureus

V8 protease digests; these were performed at 37® C for 18 hr in ammonium bicarbonate buffer, pH 7.5 containing 2M urea. Even in this denaturing buffer, not all of the subunits from HPLC were solubilised; hence the material available for peptide mapping was limited. Peptides were separated according to hydrophobicity on a C^g reverse-phase HPLC column using a gradient of acetonitrile (Figs. 3.7 and 3.8). Peptide maps for the H-subunits of types A and B CAM-BoNT (Fig. 3.8) and the H- and L-subunits from CAM-BoNT (B) (Fig. 3.7) were aligned on the same axes for easy visual comparison. Under the conditions described for elution of the column, similarities in the relative hydrophobicity of

some peptides from the aforementioned pairs of subunits were apparent.

However, the small amounts of material present in each of these peaks

( ^CO.lnmol of peptide) precluded their further analysis by either re chromatography under different conditions (e.g. pH) or accurate

determination of their amino-acid compositions. The labelling of BoNT with iodo-[I-^C] acetamide for use in this study served two purposes;

to check the alignment of eluted peptides with the fractions collected

and to indicate the position of peptides containing half- cystine

residues. A few similarities in the distribution of [^C]-radio-

activity in peptide maps of H- and L-subunits from CAM-BoNT (B) were

apparent (data not shown); however, the significance of this finding

remains unclear because the radioactive content of these peaks was very

low. Owing to the much decreased recoveries of protein following

dialysis against acetic acid, drying down and resuspension, insufficient ♦ 4 01T 206nm i. .) ee re udrvcu oe ^g n rssedd in0.1M resuspended and P^Og over vacuum under dried 3.5) were Fig. HHO cnann M ra 10l. ape ee dutd o H 7.6 pH to adjusted were Samples (150ul). urea 2M containing NH^HCO^ lmn fatos ee olce a .mn nevl. le trace, arrows Blue Vertical intervals. 0.5min at collected were fractions ml/min; -uui fo tp B type from L-subunit qiirtd ih .%tilooctc cd Ppie wr eue i a in eluted were Peptides column HPLC reverse-phase acid. trifluoroacetic 0.1% with equilibrated a to applied were digests Protein 37'C. and digested with Staphylococcus aureus V aureus Staphylococcus with digested and CAM-BoNT type B. type CAM-BoNT i. . RvrepaeHL ppiemp o oyetds rprd from prepared polypeptides of maps peptide HPLC Reverse-phase 3.7 Fig. linear gradient of acetonitrile (0-50%) over 1 hr at a flow rate of 1.5 of rate flow aat hr 1 over (0-50%) acetonitrile of gradient linear iia oiin i te ctntie gradient. acetonitrile the in positions similar oyetds rm ih rsue emain hoaorpy (see chromatography permeation pressure high from Polypeptides ( ^ ) ^ ( eoe etds f h to uuis ltn at eluting subunits two the of peptides denote 0 8 T rd rc, -uui rm ye BoNT. B type from H-subunit trace, red NT; 114 - 114 8 rtae 2u) o 8 r at hr18 for (20ug) protease ie [mini Time - 115

Fig. 3.8 Comparison between reverse-phase HPLC peptide maps of

alkylated H-subunits from types A and B neurotoxins.

Protease digestion and chromatography was performed as described

in the legend to Fig. 3.7. Blue trace, H-subunit from type A BoNT; red

trace, H-subunit from type B BoNT. Vertical arrows ( ) denote peptides

of the two subunits eluting at similar positions in the acetonitrile

gradient. material was available to produce a meaningful map of peptides from the

L-subunit of type A CAM-BoNT.

3.4 DISCUSSION

The overall similarities in molecular size of the botulinum neuro toxins and their pharmacological actions have been known for many years

(Sugiyama, 1980; Simpson, 1981a). However, minimal data is available on the further characterisation of these toxins' chemical composition and structure. In this chapter amino-acid compositions of subunits prepared from BoNT (A and B) are described for the first time together with peptide mapping studies that reveal some expected and unexpected similarities in subunit structure.

In Chapter 2, polypeptides from BoNT (A) were prepared using an ion-exchange chromatography procedure that was published recently

(Kozaki et al_., 1981). Although an essentially pure preparation of the

H-subunit was obtained by this method the L-subunit had a considerable degree of contamination. A seemingly straightforward method of preparing these subunits in a pure form, considering that retention of neurotoxicity was inessential for the studies in hand, was to use preparative SDS-PAGE. Polypeptides obtained in this manner were used for amino-acid analysis; great care was taken to avoid background contamination arising from glassware, hands, acrylamide gels and dialysis membranes (see Brown and Howard, 1983). Conditions that may have affected subsequent elution of proteins from gels and/or accurate estimation of amino-acid residues (e.g. protein staining in acidic conditions) were also avoided. The total recovery of polypeptides by electroelution of gels was generally 30-40% of that applied to the SDS- acrylamide gel; this was only slightly lower than recoveries observed when subunits were prepared using ion-exchange chromatography (see - 117 -

Chapter 2) but the purity was much improved. The particular method of

electroelution used (Fig. 3.1) was chosen for two reasons; firstly the

area of dialysis membrane exposed to the protein is much reduced (a

single circular piece of membrane about 6mm in diameter) thus reducing

* non-specific absorption and avoiding the risk of significant background

contamination of amino-acids from the dialysis membrane (Brown and

Howard, 1983). In addition, this apparatus concentrates the sample in

the region of the dialysis membrane in the anode arm of the sample * holder. On SDS-PAGE (non-reducing conditions), only the L-subunit from

type A BoNT was found to contain an extraneous band at a higher

molecular weight; this is thought to be due to reoxidation of

« half-cystine residues to from intermolecular disulphide bridges, hence

the of the contaminating band is approximately twice that of the

L-subunit. Such reoxidation is also thought to be the main contributory

factor to the high molecular weight bands observed when electrophoretic m peptide mapping was performed on subunits separated from native

(non-alkylated) BoNT (A and B).

It has long been established that some bacterial and plant toxins m are comprised of distinct subunits exhibiting different functional

activities (Collier, 1975; van Heyningen, 1976; Johnson, 1982; Olsnes

and Pi hi, 1982). A report by Kozaki (1979) suggests that the poly

peptides of BoMT (B) also have different functions; one being involved

in toxin binding and the second chain with some other, as yet, undefined

function, possibly in the lytic phase of the toxin's action. If it is

♦ believed that the neurotoxin binds to its specific neuronal acceptors) through its H-subunit enabling the other subunit to penetrate the hydro-

phobic domains of the membrane (see Chapter 6), one might expect a

significantly higher proportion of hydrophobic residues to be found in

the L-subunit to facilitate such penetration. Also, it has been

reported that on reduction of type A BoMT in low salt solutions, the - 118 -

L-subunit is preferentially precipitated (Krysinski and Sugiyama, 1980) suggesting a more inherent hydrophobic nature of this polypeptide.

These differences in physical properties and function of the subunits ought to be reflected in their basic chemical structure (i.e. their amino-acid composition and sequence). The amino-acid compositions of subunits obtained by preparative SDS-PAGE were compared to see if there were any significant differences between individual polypeptides.

Surprisingly perhaps, the proportion of the different classes of amino- acids present in the subunits from type A BoNT were almost identical.

The composition of subunits from type B BoNT was also similar except for a slightly higher hydrophobic amino-acid content of the L-subunit compared to that of the H-subunit; whether this difference is significant or not remains to be established. The hydrophobic nature of the L-subunit may be accounted for by differences in the primary sequence of the protein resulting in the exposure of hydrophobic amino-acid side chains, without necessarily increasing the relative proportions of these residues present. These hydrophobic residues may be protected from a polar environment by the tertiary structure of the intact neurotoxin; reduction of disulphide bonds may result in exposure of such residues to the hydrophilic environment and thus facilitate toxin aggregation and hence precipitation. It may be noted here that neither of the subunits of other bacterial toxins, such as diphtheria

(Delange et al_., 1979), tetanus (Taylor et al., 1983) and cholera (Ward and van Heyningen, 1981) toxins, appear to have a pronounced hydrophobicity. However, in the case of cholera toxin, only the

A^-peptide is thought to penetrate deeply into the lipid bilayer

(Wisnieski and Bramhall, 1981). It has been suggested that the A2 subunit of this toxin may act analogously to leader sequence peptides

(which are thought to facilitate export of intracellular proteins) in the entry of the A^-subunit into the cell (Waksman et al_., 1980). A compositional index, SAn, was calculated for various pairs of subunits from types A and B BoNT; the values obtained suggest that similarities exist between the two H-subunits and possibly between both

L-subunits, as might be expected. In addition, when a comparison was made between the H- and L-subunits of either BoNT (A) or BoNT (B) there was also a weak indication that the proteins may be related. The reliability of this "weak test” of relatedness, in which S A n < 0 . 9 3 N , is illustrated from the fact that out of 140 comparisons between probably unrelated proteins the 'weak test' gave a spurious indication of relatedness in only 16 cases, whereas it correctly indicated relatedness in 22 out of 23 comparisons between related proteins studied at the same time" (Cornish-Bowden, 1983). However, the use of the index

S A n when |NX-Ny |>18, as often is the case in comparing proteins of large Mr, will tend to overestimate sequence differences and thus proteins classified as being weakly related may in fact have much greater sequence homology.

In contrast to the above findings for subunits from types A and B

BoNT , Syuto and Kubo (1981) report that the polypeptides of type C BoNT possess different amino acid compositions; the H-subunit containing mostly acidic and neutral amino-acids whereas the L-subunit contained more of the basic residues. However, these differences may not be significant; the value for SAn, using half thenumber of residues in the H-subunit and the number of residues in the L-subunit of BoNT (C), was calculated as 81 (N=436). This is much less than 0.42N and therefore the conclusion ought to be that the polypeptides are very much related. Interestingly, a similar result has been reported for tetanus toxin, a protein of similar size and subunit structure to BoNT; S A n was calculated to be 60 (N = 437) which is a l s o < 0 . 4 2 N (Taylor et al.,

1983). Using SAn, no similarities between the H-subunit of tetanus toxin and the H-subunit of BoNT (A, B or C) were evident, although there is a weak indication of relatedness between the L-subunit of tetanus toxin and the corresponding polypeptides froin BoNT (A, B and C); in these latter cases 0.42N

481, 445 and 436 respectively). Tetanus toxin binds to acceptor molecules through its H-subunit (van Heyningen, 1976) as has also been suggested for type B BoNT (Kozaki, 1979). Differences in protein structure between the H-subunits of tetanus and botulinum neurotoxins would have a bearing upon their interactions with specific acceptor(s).

Furthermore if the L-subunit is the sole source of these toxins' pharma cological activities, bearing in mind the similarities in action of tetanus and botulinum toxins (Habermann, 1981; Sugiyama, 1980), one might expect them to show at least some structural homologies; the data presented above support this assumption.

To examine more directly structural homologies between the sub units of BoNT, one-dimensional peptide mapping was performed on poly peptides obtained from native and types A and B CAM-BoNT. Peptide mapping of subunits by SDS-PA6E gave similar results whether native- or

CAM-BoNT was used, although the modified toxin gave more satisfactory patterns. Maps of the H-subunits from CAM-BoNT (A and B) shared several bands of similar electrophoretic mobility as did those of both L- subunits. Moreover, similarities were apparent between the H- and L- subunits of the same type of CAM-BoNT. This technique of peptide mapping accentuates differences in primary protein structure and thus even a small degree of correspondence between maps may be of significance in terms of the original protein sequence. The aforementioned similarities were confirmed using reverse-phase HPLC to separate peptides obtained from total Staph, aureus V8 protease digests of toxin subunits.

~~An interesting hypothesis brought to attention by Taylor etal_.~~

(1983), to account for their findings with tetanus toxin, is that the intact toxin has arisen from duplication of an ancestral gene coding for a single protein of xSOOOO. This would also account for the similarities in structure and composition noted with BoNT (A, B and C) above. In this context it is pertinent to recall that the H-subunit of

~~BoNT is rather more resistant to proteolysis than the L-subunit, the first notable cleavage of the H-polypeptide being about halfway along the give two proteins (H^ and H^) of similar M (n.50000)~~

~~(Sugiyama, 1980). Also limited treatment of tetanus toxin with papain results in the cleavage of the H-subunit at its midpoint (Helting and~~

~~Zwisler, 1977). However, if a single ancestral gene coding for a protein of 50000 was involved, substantial evolutionary changes must have occurred to provide each of the subunits of various types of~~

~~BoNT with their individual immunological characteristics (see Chapter 2;~~

~~Kozaki and Sakaguchi, 1975; Krysinski and Sugiyama, 1980; Syuto and~~

~~Kubo, 1981).~~

The interpretation of observations from the structural studies reported herein must be made with caution; protein bands on an SDS- acrylamide gel or proteins eluting as one peak from a reverse-phase HPLC column may easily be heterogeneous with regard to their peptide content.

Ideally one would wish to check for homogeneity of a particular peptide by rechromatography or electrophoresis under different conditions followed by N-terminal analysis to confirm the presence of one peptide sequence. Only with such precautions could one confidently relate similarities in peptide maps to homologous protein sequences.

Unfortunately, the amount of peptide in each peak eluted from the reverse-phase chromatography column was insufficient to obtain a meaningful amino-acid analysis, which hopefully might help substantiate the similar composition of these peaks. However, it is interesting that there is some indication of homology in peptides containing half cystine residues from the H- and L-subunits of type 3 BoMT; a previous report (DasGupta, 1981) has suggested that there may also be homology in the positions of half-cystine residues between different neurotoxin types. - 122 -

~~The subunits used for reverse-phase HPLC peptide mapping were~~

~~successfully prepared from CAM-BoNT (A and B) by high pressure~~

~~permeation chromatography under denaturing conditions. This method of~~

~~subunit preparation has advantages over previously reported methods; the~~

* time required to achieve the separation is about 30 min compared with

~~many hours with an ion-exchange procedure (Kozaki et al_., 1981) or~~

~~longer by preparative SDS-PAGE. In addition, high recoveries of protein~~

~~(e.g. 70-80%) may still be achievable after rechromatography of eluted * peaks in order to achieve complete sample purity. This method of sub~~

~~unit preparation may easily be applied to the other types of dichain~~

~~BoNT and tetanus toxin; to date different methods have had to be~~

* employed for the preparation of polypeptides from these various types of

~~neurotoxin (Matsuda and Yoneda, 1975; Kozaki et a K , 1977; Krysinski~~

~~and Sugiyama, 1980; Kozaki et al^., 1981; Syuto and Kubo, 1981).~~

• *

* CHAPTER 4

~~RADIQIQDINATIQN OF TYPES A AND B BONT:~~

~~THEIR SPECIFIC INTERACTIONS WITH CEREBROCORTICAL SYNAPTOSOMES 4.1 INTRODUCTION~~

The electrophysiological effects of haemagglutinin-neurotoxin complexes from Cl. botulinum on the NMJ have been well characterised; these complexes act irreversibly at the presynaptic motor nerve terminal to inhibit specifically the release of ACh (Harris and Miledi, 1971;

Cull-Candy et al_., 1976a* Mackenzie et a K , 1982). Such apparent specificity in the action of these neurotoxins, at least at the NMJ, may allow them to be used as probes for components unique to cholinergic presynaptic membranes and possibly for an intra-terminal target involved directly in the mechanism(s) of neurotransmitter release.

Early studies on botulinum toxin binding, in vitro, to neuronal tissue employed ferritin or fluorescein-labelled botulinum toxin- haemagglutinin complexes (Zacks et a K , 1962; 1968). These studies revealed preferential binding to areas of nerve muscle preparations thought to represent the terminal regions of the nerve. This group

found similar patterns of labelling using unlabelled toxin complex and fluorescein-labelled antitoxin (Zacks et al_., 1968). Interaction of the toxin with mouse tissues was reportedly selective for motor endplates since they failed to observe binding to heart, lung, liver, spleen, kidney or brain tissue.

~~Crystal!ir reparations of type A neurotoxin-haemagglutinin 3 complex, tritiated by propionylation with N-succinimidyl [2,3- H] propionate, were found to bind with high affinity to rat cerebrocortical~~

~~synaptosomes (Dolly et al^., 1982). However, the efficiency of~~

galactose, an inhibitor of haemagglutinin, in preventing the major part of this binding and the ability of toxin complexes to agglutinate red blood cells suggest a dominant role of the more abundant (relative to

~~the neurotoxin moiety of the protein complex) haemagglutinin components.~~

In view of this inherent complication of heamagglutinin binding and the large molecular weight of such complexes, they are unsuitable for use as specific neurochemical probes. Hence, the significance of the above report that fluroescent or ferritin-labelled derivatives of such complexes bind selectively to the NMJ remains to be established; also, such reagents cannot be used for quantification of acceptor sites present. Hence, preparation of a neurotoxic, radiolabel led derivative of the pure neurotoxin is a prerequisite for exploiting the usefulness of this valuable neurochemical tool.

Owing to the apparent sensitivity of the neurotoxin moiety to radiolabelling, workers have previously labelled neurotoxin- 125 haemagglutinin complexes with I-iodine and subsequently isolated a labelled neurotoxic component from this by gel filtration (Habermann,

1974; Wiegand et. al_., 1976). Such a procedure results in the majority of the label being incorporated into the haemagglutinin moiety. Hence the neurotoxic component, in addition to being only partially purified, is of very low specific radioactivity; owing to the extreme potency of this neurotoxin such derivatives are thus of very limited use. Attempts to date have failed to radioiodinate BoNT to the measureable (Kozaki,

~~1979) and high levels (Kitamura, 1976) of specific radioactivity necessary for its sensitive detection without appreciable losses in stability and biological activity (reviewed by Simpson, 1981).~~

In this study a method was developed for the radiolabelling of 125 type A BoNT with I-iodine to high specific radioactivity with retention of its neurotoxicity and ability to bind specifically at motor nerve terminals, as shown by light-microscope autoradiography (Dolly et al., 1981,1982). The specificity of such binding to the presynaptic membrane at the NMJ was shown recently using electron-microscope auto radiography (Black et a K , 1983; Dolly et al_., 1984a). This method of radiolabelling was also found to be suitable for the iodination of type

~~B BoNT. Owing to the unsuitable nature of the neuromuscular junction - 126 -~~

~~for detailed biochemical investigation, rat brain synaptosomes were used~~

~~as a model to study the specific interaction(s) of botulinum neurotoxins~~

~~with their neuronal acceptor(s). Acceptors for I-labelled~~

~~botulinum neurotoxin, types A and B, on rat brain synaptosomes have been~~

~~characterised and are shown to be located preferentially on synaptosomal~~

~~plasma membranes.~~

~~4.2 METHODS~~

~~4.2.1 Radiolabelling of botulinum neurotoxins. BoNT was routinely 125 radiolabelled with I-iodine using a modification of the~~

~~chloramine-T method (Greenwood et a!., 1963). BoNT (A) (100-200ug) in a~~

~~maximum volume of 200ul was added to 5mCi (50ul) carrier-free Ma I~~

~~(Amersham International, Amersham) and the reaction was initiated by the~~

~~addition of chloramine-T (5ul; final concentration, 0.22mM in lOOmM~~

~~sodium phosphate buffer, pH 7.4). The reaction was quenched after 40s~~

~~by the addition of an excess of L-tyrosine (50ul; lmg/ml in lOOmM sodium~~

~~phosphate buffer, pH 7.4, containing 150mM NaCl). I-Labelled type~~

~~A BoNT [ I-BoNT (A)] was separated from tyrosine and free iodine by~~

~~gel filtration on a Sephadex G-25 superfine column (5 x 1.5cm), pre~~

~~equilibrated with lOOmM sodium phosphate buffer, pH 7.4, containing~~

~~150mM NaCl. To ensure stability of the neurotoxin in dilute solution,~~

~~it was diluted with 2.5% (w/v) gelatin to a final concentration of 0.25%~~

~~(w/v), after removal of aliquots for determination of protein and radio~~

~~activity. The amount of free radiolabel present in a labelled-toxin~~

~~sample (5-10ul) was determined by precipitation with trichloroacetic~~

~~acid, 10% (v/v), using bovine serum albumin (lOOug) as a carrier~~

~~protein. Type B BoNT, in its native, partially-nicked form was radio-~~

~~labelled in an identical manner to that described above for type A BoNT.~~

~~Protein and toxicity determinations were carried out as described~~

previously (see Methods 2.2.8). 4.2.2 Characterisation of 125I-BoNT (A and B). The toxins' electro phoretic mobilities and distribution of radioactivity in their subunits were determined by discontinuous SDS-gel electrophoresis (Laemmli, 1970) on a 10% polyacrylamide gel run under non-reducing and reducing (5% 2- mercaptoethanol in the sample buffer) conditions (described in Methods

2.2.5). After fixing, staining and destaining, the gels were washed extensively in deionised water prior to being dried and submitted to autoradiography. The distribution of radioactivity between the subunits was confirmed by slicing the gel (2.5mm) and V-radiation counting.

~~The immunoreactivity of the I-BoNT (A) was studied using~~

Ouchterlony double immunodiffusion. Gels (5 x 5 x 0.2cm) were prepared using 1% Agarose in lOOmM sodium phosphate buffer, pH 7.0; centre wells contained horse antibodies (5[il) raised against botulinum neurotoxin- haemagglutinin complex (type A). Aliquots of samples (5ug native BoNT or 0.2ug I-BoNT) were placed in surrounding wells and left to diffuse for 24 hr at 4°C. The gel was washed, fixed, dried (onto a glass plate), stained with Coomassie blue R-250 and destained (see

~~Methods 2.2.6) before being subjected to autoradiography.~~

~~Narrow range (pH 5-8) disc gel isoelectric focussing (IEF) of type 125 A I-BoNT under native conditions was carried out according to~~

O'Farrell (1975) as described previously (see Methods 2.2.5). Focussed gels were sliced (2mm), equilibrated in lOmM KC1 and the pH gradient determined. Radioactivity associated with gel slices was quantified by

~~^-radiation counting. The position of native toxin was determined by protein staining an unsliced track of the gel with Coomassie blue G-250~~

~~(0.075%) in 3.5% perchloric acid.~~

4.2.3 Separation of native toxin from its labelled species. Chromato- focussing (pH 6.0-7.3) was performed on I-BoNT (A) immediately following radio!abelling. Iodinated toxin was equilibrated in 0.025M - 128 -

~~imidazole-acetate buffer, pH 7.3, by gel filtration on Sephadex G-25~~

~~(superfine) and applied to a Polybuffer Exchanger 94 (PBE 94, Pharmacia)~~

~~chromatofocussing column (5.5 x 0.3cm). Following elution of unbound~~

~~radioactivity with the above buffer, a pH gradient was commenced by~~

* stepwise elution of the gel column with polybuffer 96-acetate, pH 6.0

~~(Pharmacia). Aliquots (5- 10pl) were removed from each fraction UlOpl)~~

~~and subjected to -radiation counting. The toxicity of various~~

~~fractions was determined by intraperitoneal injection into mice as * described previously. The pH gradient was measured using a pH~~

~~microelectrode.~~

~~A m on-exchange chromatography of I-BoNT (A) was performed on~~

~~♦ DEAE-Sephadex A50, equilibrated in 40mM Tris-HCl buffer, pH 7.0. Bound~~

~~toxin was eluted with a linear chloride gradient (10ml; 0-0.3M NaCl)~~

~~formed by mixing bml of 40mM iris-HCl buffer, pH 7.0 and 5ml of the~~

~~% same buffer containing 0.3M NaCl. Fractions U150pl) were collected into tubes containing 2.5% (w/v) gelatin (15pl), from which aliquots~~

~~were taken for -radiation counting, determination of toxicity and~~

~~chloride ion concentration. The latter was determined colorimetrically if using stock solutions of (A) 4% Fe (N03 )3 in 0.5M HN03 and (B)~~

~~HgtCNSlg saturated in ethanol; 0.5ml of solution B was added to the~~

~~diluted sample (0.5ml) together with 0.5ml of solution A. After lOmin~~

* at room temperature the absorbance was read at 460nm; a standard curve

~~was constructed using 0 - 2.0mM NaCl.~~

~~4.2.4 Preparation of rat cerebrocortical synaptosomes. These were~~

~~routinely purified from brains of female Sprague-Dawley rats (%200g) by~~

~~discontinuous sucrose density-gradient centrifugation (de Belleroche and~~

~~Bradford, 1977). Following dissection of the brain and removal of the~~

~~cerebellum and white matter, the cortex was homogenised in 0.32M sucrose~~

and centrifuged (800 x g, lOmin). The sediment (P^) was discarded and the supernatant centrifuged (20000 x g, 20min) to sediment the crude synaptosomal fraction (P2 ). The P2 was homogenised in 0.32M sucrose and layered (15ml per gradient) onto a discontinuous sucrose gradient consisting of 1.2M sucrose (20ml) and 0.8M sucrose (20ml). The gradients were centrifuged in a swing-out rotor for 60min at 76000 x g.

Purified synaptosomes were collected from the 0.8-1.2M sucrose interface and diluted slowly to 0.45M sucrose with ice-cold deionised water, before collection by centrifugation (54000 x g, 20 min) and resuspension in Krebs-phosphate buffer, pH 7.4; this buffer contained the following concentrations of salts (mM), NaCl, 124; KC1, 5; Na^PO^, 20;

KHgPO^, 1.2; MgSO^, 1.3; CaCl2 , 0.75 and glucose, 10. All procedures were carried out at 4°C. To examine any degradative effect of endogenous proteases, the protease inhibitors EGTA (ImM), EDTA (ImM), phenyl methyl sulphonyl fluoride [PMSF] (0.5mM), pepstatin (lOpg/ml) and benzamidine (ImM) were present in the sucrose solutions used. Lysed synaptosomes were obtained either by freeze-thawing or treatment of synaptosomes with 50mM Tris-HCl buffer, pH 8.0, for 30 min at 4°C.

~~125 4.2.5 Measurement of I-BoNT (A and B) binding to synaptosomes.~~

~~Purified synaptosomes suspended in Krebs-phosphate buffer, pH 7.4, were diluted into the same buffer containing lmg/ml bovine serum albumin.~~

Saturability of binding was ascertained by incubation of synaptosomal suspensions (0.5-0.7mg/ml) with increasing concentrations of types A or 125 B I-BoNT for 60 min at 37 C. Subsequently, suspensions were centrifuged (9000 x g, 2min) and aliquots removed from supernatants for the determination of free toxin concentrations. The synaptosomal pellets were washed twice (within 20 min) in ice-cold Krebs-phosphate buffer (containing albumin) before being submitted to -radiation counting. Non-specific binding was determined at various concentrations of ^^I-BoNT (A or B) by incubation in the presence of 100-fold molar excess of native BoNT (A or B) over the highest concentration of 125 I-BoNT used; it was subtracted from the total to give the amount

~~specifically bound.~~

~~Initial rates of toxin association with its binding site(s) at 4°C 1 oc were determined by using 0.6-6.0nM type A or B I-BoNT and 0.5-1.0~~

~~mg/ml of synaptosomal suspension. Binding was terminated at various~~

~~intervals by removal of aliquots (50-100ug protein) from a batch~~

~~incubation, dilution with ice-cold buffer and centrifugation.~~

~~Sedimented membranes were washed once (within 5-8 min at 4°C , to~~

~~minimise dissociation) with the above buffer before ^-radiation~~

~~counting. Non-specific binding was calculated as detailed above. Rates 125 of dissociation of type A or B I-BoNT from synaptosomal membranes~~

~~were studied at 4°C, after preincubation of synaptosomes (0.6mg/ml) in 125 a batch with type A or B I-BoNT for 40 min at 37 C. The suspension~~

~~was cooled on ice for 10 min (from which aliquots were taken for zero~~

~~time points) before dissociation was initiated by dilution with ice-cold~~

~~buffer (20-25 fold excess volume) in the absence or presence of native~~

~~BoNT (A or B); under the latter conditions, a 100-fold molar excess of 125 BoNT over the final concentration of I-BoNT was used. At various~~

~~time intervals aliquots of the suspensions were removed and centrifuged;~~

~~synaptosomal pellets were washed once (within 6-8 mins at 4°C) before~~

~~binding was quantified by V-radiation counting. Non-specific binding~~

~~was determined in the presence of an excess of native BoMT throughout.~~

~~4.2.6 Effect of temperature and pH on synaptosomal binding of type A 125 I-BoNT. Synaptosomes (50-100ug protein) were incubated for 40 min~~

~~at either a series of temperatures (4-37°C) or pH (at 37®C) values with 125 " I - BoNT (A), 2nM or InM toxin respectively. Binding was terminated~~

~~by dilution with ice-cold buffer and centrifugation. Synaptosomal~~

~~pellets were processed and specific binding calculated from total and - 131 -~~

~~non-specific binding as detailed above. Krebs-phosphate buffer~~

~~containing albumin was adjusted to the appropriate pH by addition of~~

~~dilute hydrochloric acid or sodium hydroxide solution; all synaptosomal~~

~~suspensions and toxin dilutions were in buffer of the required pH.~~

~~4.2.7 Electron-microscope autoradiography of synaptosomes labelled~~

~~with I-BoNT (A). Synaptosomes were purified as described above~~

~~except that Ficoll (Pharmacia) density gradients were used (Cotman and~~

~~Matthews, 1971). Discontinuous gradients were prepared with 20ml of 13%~~

~~(w/w) Ficoll, 20ml of 8% (w/w) Ficoll and 15ml of crude synaptosomes~~

~~(Pg, see above) in 0.32M sucrose. All Ficoll and sucrose solutions~~

* were prepared in 2mM HEPES buffer, pH 7.5. Synaptosomal suspensions

~~were incubated with InM type A I-BoNT for 40 min at 37°C, binding~~

was terminated by dilution and centrifugation as described elsewhere. 125 Control samples were incubated with type A I-BoNT in the presence ♦ of 10“^M native BoNT (A). After washing twice in ice-cold buffer,

~~synaptosomal pellets were fixed in Krebs-phosphate buffer, pH 7.4,~~

~~containing 2% glutaraldehyde (60 min at 22°C). Fixed samples were~~

~~washed with Krebs-phosphate buffer prior to treatment with 1% OsO^ (60~~

~~min, 22°C); samples were then washed in buffer and following de~~

~~hydration in ethanol and embedding in Spurr's resin (Spurr, 1969), pale~~

~~gold sections were cut on an LKB ultramicrotome and prepared for~~

~~electron-microscope autoradiography by the 'flat substrate method' of~~

~~Sal peter and Bachmann (1972); they were covered in a monolayer of~~

~~Ilford L4 emulsion by the dipping technique. After 3 weeks exposure,~~

~~specimens were placed in Kodak D-19 developer (2 min, 22°C), stained~~

~~with 12% uranyl acetate and examined in a Hitachi electron-microscope. - 132 -~~

~~Fig. 4.1 Sephadex G-25 chromatography of type A 125I-Bo NT.~~

~~1ne 125 I-BoNT was separated from tyrosine and free I by gel~~

~~filtration on a column of Sephadex G-25 superfine (5 x 1.5cm)~~

~~equilibrated with lOOmM sodium phosphate buffer, pH 7.4, containing~~

~~150mM NaCl. Eluted fractions (%200pl) were collected and aliquots (3ul)~~

~~taken for y -radiation counting. The arrow (\JO indicates the bed~~

~~volume of the column; note that elution of free iodine is delayed; this~~

is probably due to non-specific interactions. 9 0 4 # * *

~~125 Table 4.1 Conditions for I-iodination of type A BoNT using the chloramine-T method.~~

~~A selection of conditions tested for the radiolabelling of BoNT are shown, where the reaction volume was 0. 1-0.7ml.~~

~~125I-Bo NT~~

~~Specific Toxicity ^ I - l o d i n e radioactivity *Speci fic BoNT (m M) Chloramine-T (mM) (liM) (Ci/mmol) (mouse LDcn/mg) Recovery (%) binding~~

~~10.66 1.88 6.41 400 6.0xl06 4.0 0~~

~~3.74 0.87 5.95 3751 2.5x10s 1.2 0~~

~~2.69 0.86 5.88 2209 3.6xl07 45.0 80~~

~~2.16 0.56 3.79 239 7.6x107 84.0 60~~

~~2.27 0.53 3.98 140 2.2xl07 53.0 65 133~~

~~3.43 0.22 7.27 854 2.0x107 25.0 60~~

~~8.24 0.29 9.62 1430 9.7xl07 53.9 88~~

~~9.34 0.22 16.34 700 3.3x108 83.5 95~~

~~9.22 0.22 16.13 712 3.4xl08 85.0 93~~

~~8.93 0.29 15.63 1532 1.2xl08 60.0 85~~

~~6.80 0.22 23.81 1750 1.2X108 60.0 92~~

* Specific bindi ng was determined as the percent of radiolabelled toxin displaced from rat cerebrocortical synaptosome

~~suspensions in a routine competition assay in the presence of 10”9M 125I-BoNT (A) and 10“6M native BoNT (A). Specific~~

~~binding was retained for 2-4 weeks. 4.3 RESULTS~~

~~125 4.3.1 Properties of type A I-BoNT. Homogeneous BoNT (A) was radio- 125 labelled with [ I]-iodine using a modification of the chloramine-T~~

~~method (Greenwood et al^, 1963). After separation of labelled protein 125 from free I and tyrosine by Sephadex G-25 gel filtration (Fig.~~

~~4.1), the specific activity was accurately determined and found to be at~~

~~a high level (e.g. 1750 Ci/mmol). When the optimal conditions were used~~

~~the biological activity of the resultant preparation was high (e.g. o 1.2 x 10° mouse LD^/mg; see Table 4.1) as shown by serial, intra-~~

~~peritoneal injections into mice. Also, the biological activity was~~

~~confirmed by autoradiographic localisation of specific binding sites for 125 I-BoNT at murine neuromuscular junctions (Dolly et a K , 1982).~~

~~The successful radioiodination of BoNT was accomplished by varying~~

~~the reaction conditions and concentrations of reactants (Table 4.1).~~

BoNT of relatively low specific toxicity (2.2 x 10^ mouse LD^/mg) was rather sensitive to the iodination procedure, particularly when high concentrations of chloramine-T were used; less than 2% of the initial 125 toxicity was recovered, but high incorporation of I was achievable

~~Q (3751 Ci/mmol). BoNT of high specific activity (3 x 10 mouse~~

~~LDgQ/mg) treated in an identical manner, was radio!abelled to a~~

~~similar level and with higher retention [13%) of its biological activity~~

(data not given in Table). To preserve an even larger fraction of biological activity during labelling, lower concentrations of chloramine-T (0.22-0.29mM final concentration) were tested. Under these optimal conditions and using toxin of high specific neurotoxicity

~~(2-4 x 10® mouse LD^/mg protein), ^®I-BoNT preparations retained high levels (60-85%) of biological activity (Table 1) and were usable~~

for 2-4 weeks; on more prolonged storage the level of nonspecific binding to brain synaptosomes increased from as little as 5% to more 125 Fig. 4.2 Autoradiographic analysis of I-BoNT (A) following 5D5-

~~PA6E.~~

~~125 I-iodinated samples were applied to an acrylamide slab gel~~

~~(10%) in the presence (tracks 4 and5) or absence (tracks 1 , 2 and3) of~~

~~2-mercaptoethanol. Following electrophoresis, the gel was stained with~~

~~Coomassie blue R-250, destained, dried and submitted to autoradiography.~~

Molecular weights were determined by protein staining of standard markers; native type A BoNT was run in adjacent tracks under non reducing and reducing conditions to compare mobilities of the labelled and unlabelled species. Arrows indicate position of native neurotoxin or its constituent polypeptides. - 136

~~125 Fig. 4.3 Inununoreactivity of native and I-BoNT (A).~~

♦

* ’

~~125 Samples (5ug native type A BoNT, 0.5pg type A I-BoNT) were~~

~~placed in peripheral wells and horse anti-(neurotoxin-haemagglutinin~~

~~complex) antibody (1000 units/ml; 1 unit neutralises 104 mouse~~

~~LD,-0) in the central well. These were left to diffuse for 24 hr at~~

~~4°C. The gel was washed, fixed, dried, stained with Coomassie blue~~

~~R-250 and destained before being subjected to autoradiography, (a) 0 125 I-BoNT (autoradiogram); (b) native BoNT (protein stained); (c)~~

~~precipitin lines from (a) and (b) superimposed by placing the auto~~

~~radiogram of the dried gel over the protein stained glass plate. 0 - 1 3 7 -~~

~~Fig. 4.4. Isoelectric focussing of type A 125I-BoNT.~~

~~pH ♦~~

♦

~~125 The isoelectric point of I-BoNT was determined by narrow~~

~~range (pH 5-8) disc gel isoelectric focussing with lOmM at the~~

~~anode and 20mM NaOH at the cathode. Focussing was performed at 400Y~~

~~overnight at 4°C. The gel was sliced, equilibrated in lOmM KC1 and the~~

~~pH gradient determined (O). Radioactivity associated with the slices~~

~~(•) was determined by Y-radiation counting. The labelled toxin showed a~~

~~single peak of radioactivity with a pi = 6.9 ± 0.3 (n=3); the small~~

~~amount of radioactivity present at the basic end of the gel is due to~~

non-specific adsorption of toxin upon its application. than 50% (see below). Stability appeared to be dependent on the specific toxicity of the native neurotoxin, with less active preparations becoming inactive more quickly.

~~The majority (90-97%) of the radioactivity in ^I-BoNT i pc preparations was acid-precipitable. On SDS-electrophoresis, I-~~

~~BoNT gave a single radioactive band with an electrophoretic mobility similar to that of the unlabelled neurotoxin (Mr of 138000); on reduction, two radioactive species were observed (Mr of 91000 and~~

55000) corresponding to subunits of the native toxin (Fig; 4.2). The distribution of label in these subunits was observed to be proportionate approximately to their protein content when the gel slices were subjected to ^-counting.

On Ouchterlony double-immunodiffusion gels the single precipitin line produced by I-BoMT (visualised by autoradiography, Fig. 4.3A) coalesced (Fig. 4.3A) with the lines from native toxin (obtained by protein staining, Fig. 4.3B), indicating that the toxin had retained its 125 immunoreactivity after I-iodination. Using narrow range (pH 5-8) 125 disc gel isoelectric-focussing (Fig. 4.4), I-BoNT gave a single peak of radioactivity which was found to have an isoelectric point of

~~6.9 (SE ± 0.3, n = 3).~~

4.3.2 Separation of native toxin from its labelled species. Separation of unlabelled from radiolabelled toxin species was attempted by 125 chromatofocussing and ion-exchange chromatography. Type A I-BoNT chromatofocussed (pH 7.3-6.0) on a column of polybuffer exchanger 94 at

25°C showed the major eluted peak to have a pi of 6.7 and a minor component eluting at pH 6.5 (Fig. 4.5). The peak of radioactivity eluting in the column void volume was mainly contributed by free I- 125 125 iodine or I-tyrosine, although a small amount of I-BoNT was also present (shown by SDS-PAGE). - 139 -

~~Fig. 4.5 Chromatofocussing of type A 125I-BoNT.~~

~~% Attempts to separate native and radiolabelled toxin species were~~

~~made by chromatofocussing (pH 6.0-7.3) of I-BoNT (A) on Polybuffer~~

~~Exchanger 94 (PBE94, Pharmacia) at 25°C. ^ I - B o N T was equilibrated~~

~~in 25mM imidazole-acetate buffer, pH 7.3 by gel filtration on a column * of Sephadex-G25 (superfine) immediately following radioiodination.~~

~~Equilibrated toxin was applied to a PBE94 column in the same buffer and~~

~~bound toxin was eluted in a pH gradient (O) initiated by the addition of « polybuffer 96-acetate, pH 6.0. Aliquots (5pl) were removed from each~~

~~fraction (^lOOpl) and submitted to y-radiation counting (•). Two radio~~

~~active peaks were observed in the pH gradient with pl=6.7 and 6.5; the~~

~~radioactivity in the column void volume is contributed mainly by free~~

~~^ 5I-iodine and/or ^ 5I-tyrosine. It is not certain whether the second peak, eluted in the pH~~

~~gradient, is due to the presence of a di- or higher-labelled toxin~~

~~species or to microheterogeneity in the native toxin. Owing to the~~

~~lower specific radioactivity (510 Ci/mmol) of the labelled preparation~~

~~used in this particular experiment and evidence from narrow range iso electric focussing of native toxin (which showed two bands separated by~~

~~< 0 . 0 5 pH units - unpublished data), it is possible that different mono- labelled species and microheterogeneity in the native toxin both con~~

~~tribute to the presence of two radioactive peaks. The presence of di-~~

~~or higher-labelled toxin species could not be dismissed or confirmed by~~

~~expressing the radioactivity of the eluted material per mouse LD^q~~

~~owing to a much decreased toxicity of the eluate; likewise, the~~

~~position of native BoNT could not be confirmed. This was shown to be~~

~~due to instability of the neurotoxin in the elution buffer used; toxin~~

~~stored overnight in polybuffer 96-acetate, pH 6.0, exhibited only 20% of~~

~~its original toxicity when compared to similar treatment in 0.1M sodium~~

~~phosphate buffer, pH 7.5. Therefore, owing to the unsatisfactory~~

~~resolution of the eluted peaks and the instability of the neurotoxin in~~

~~the elution buffer, this method was found unsuitable for the separation of labelled and unlabelled toxin species.~~

~~A column of DEAE-Sephadex A50 equilibrated in 40mM Tris-HCl 125 buffer, pH 7.0, bound type A I-BoNT, whilst free radioiodine was~~

~~eluted in the column washthrough (Fig. 4.6). Bound toxin was eluted in a~~

~~single broad peak of radioactivity by the application of 75-125mM NaCl.~~

~~This eluted toxin exhibited biological activity and represented about~~

~~20% of the total radioactivity applied to the column. The toxicity~~

~~content of fractions 70, 80 and 90 were similar, indicating that native~~

~~toxin may be eluted from the ion-exchange column just prior to the~~

~~labelled species. However, such broadness of the eluted peak and low~~

~~recovery of applied toxin did not warrant further development or routine - 141 -~~

~~125 Fig. 4.6 Anion-exchange chromatography of I-BoNT (A).~~

~~Freshly radioiodinated BoNT was equilibrated in 40mM Tris-HCl~~

~~buffer, pH 7.0, by Sephadex G-25 gel filtration and applied to a column 4| of DEAE-Sephadex A50 (5.5 x 0.3cm) likewise equilibrated. After washing~~

~~through any unbound protein or free iodine, the column was eluted with a~~

~~linear NaCl gradient (0-0.3M). Fractions (%150ul) were collected in~~

* tubes containing 0.25% (w/v) gelatin (15ul). Radioactivity in each

~~fraction (•) was quantified by y-radiation counting and respective~~

~~chloride ion concentrations (o) were determined spectrophotometrically~~

~~(see Methods 4.2.3). One broad peak of radioactivity was eluted with * 75-125mM NaCl.~~

~~4 I-Io d in e Id pm) * * * acton n er be m nu n tio c ra F * * * * 09 H* cr» N> h-» ■ts I I - 143 -~~

~~Fig. 4.7 Equilibrium binding of type A 125I-BoNT to purified rat~~

~~cerebrocortical synaptosomes.~~

~~Synaptosome suspensions (50-150ug/incubation) were incubated with~~

~~increasing concentrations of 125I-BoNT (0.1-21nM) at 37°C. After 60~~

~~min, suspensions were centrifuged and aliquots removed for quantitation~~

~~of free toxin concentrations. Following removal of excess supernatant,~~

~~synaptosomal pellets were washed twice at 4°C before Y -radiation~~

~~counting. The Scatchard plot shown for the specific binding (see~~

~~insert) was biphasic, representable of high-affinity (KQ = 0.6nM;~~

* Bmax = 60fmol/mg protein) and low-affinity (KQ>25nM) components, as

~~shown by graphical analysis. Insert: specific binding (•) was~~

~~calculated by subtracting non-specific binding (▼), determined in the~~

~~presence of 100-fold molar excess of native BoNT over the highest ® 1 oc concentration of 1 I-BoNT used, from total binding (o)-~~

♦

* ♦ ♦ ♦ ♦ « ,§TI L'*i cl) u> o o o - tjn o m - Bound toxin/free toxin (pmol.mg“VnM ) O

~~Bound toxin (pmol/mg protein) - 145 -~~

~~use of this method for the separation of unlabelled from labelled~~

~~neurotoxin.~~

~~IOC 4.3.3 Saturable binding of I-BoNT (A) to rat cerebrocortical~~

* synaptosomes. Purified rat brain synaptosomes were used as a model to

~~facilitate investigations on the nature (see Chapter 5) and kinetics of~~

~~the binding sites for BoNT in the central nervous system which are not~~

~~presently possible at the NMJ. On incubation of synaptosome suspensions * with increasing amounts of I-BoNT (0.1- 21nM) a defined amount of~~

~~binding was observed; its saturability was demonstrated by the ability~~

~~of 100-fold excess of native toxin to prevent the binding of labelled ♦ toxin (Fig. 4.7, insert). Presentation of this data on a Scatchard plot~~

~~(Fig. 4.7) showed there was heterogeneity in the binding. Graphical~~

~~analysis of the curve (Rosenthal, 1967) showed a low content of sites~~

~~♦ with high affinity (K. = 0.6nM, Bmav = 60 fmol/mg protein), together with a much larger amount of a low-affinity component. Lysis of the~~

~~synaptosomes did not significantly affect the value observed for the 125 maximum binding of I-BoNT. « 125 Kinetics of the interaction of I-BoNT with its acceptors were~~

~~studied at relatively low concentrations of toxin with the aim of~~

~~studying mainly the high-affinity site(s); this would permit much of~~

~~the heterogeneity due to the presence of low affinity acceptors to be~~

~~avoided. Owing to the rapid association and dissociation rates at 37°C, 125 the I-BoNT binding kinetics were studied at 4 C. Interpretation of~~

* association rates in this case is best accomplished using second order

~~kinetics (see equation 4.1):~~

~~1 .in (W k+ r t Equation 4.1 R 0 -T. 0 (W 125 Fig. 4,8 Association of type A I-BoNT with acceptor sites on synaptosomes.~~

125 Association rates of type A I-BoNT binding (4nM) were studied at 4°C. (A) Aliquots of synaptosomal suspensions were removed from 125 batch incubations with I-BoNT at various time intervals, diluted with ice-cold buffer containing albumin and centrifuged. Sedimented membranes were washed once (within 6-8min) with the above buffer before

submitting to y-radiation counting; specific binding (•) was determined by subtracting non-specific (□) from total binding (o). Standard errors are given for the mean values of three determinations; in cases where error bars are not shown, errors lie within the size of the

~~points.~~

(B) Second-order rate plot of the specific binding calculated in (A); the association rate constant (k+^) for the faster-associating component present was estimated as 3.3 x 10^M." *s~*, from the second order rate equation ^— i^-.ln (T .R^/Rq .T^.) = k+1 .t, o o where RQ and TQ represent the acceptor and toxin concentration, respectively, at time zero and Rt and the acceptor and toxin concentrations at time t; k+^ is the second-order association rate constant. - 147 -

~~Fig. 4.8~~

~~15 min - 148 -~~

~~where RQ and Tq are the acceptor and toxin concentrations (M),~~

~~respectively, at zero time and, and represent the acceptor and~~

~~toxin concentrations (M) at time t (s).~~

~~Thus, for a single class of non-interacting sites, a plot of~~

* ^ .In ^ 0.Rt/R0.T^) against time(t) gives a straight line,

~~with a gradient equal to k+1 (the association rate constant). A~~

~~second order plot of the specific binding observed (Fig. 4.8A),~~

~~following incubation at periods up to 10 min with ^ I-BoNT (4nM), was * not mono-phasic (Fig. 4.88). A second-order rate plot with similar~~

~~shape was found using much lower I-BoNT concentrations (0.6nM),~~

~~suggesting that the biphasic nature of the plot, when 4nM toxin was~~

* used, does not result totally from the toxin's interaction with both

~~high- and low-affinity acceptors. From the initial rate of binding an 4 -1 -1 association rate constant of k+^ = 3.3 x 10 M .s . was~~

~~approximated. However, it must be stressed that this rate constant is * only approximate; this is owed to technical difficulties in obtaining~~

~~many accurate values for early time points ( < 2 min) using a~~

~~centrifugation assay, a contribution to the initial rate by the ♦ slower-associating component and the unavoidable dissociation of toxin~~

~~during washing .~~

~~For studies on dissociation, synaptosomes were preincubated with~~

~~♦ 4nM ^ I - B o N T at 37°C and cooled on ice (to 4°C) before measuring dis~~

~~sociation following dilution in the absence or presence of native type A~~

~~BoNT. Dissociation of specifically bound toxin was found to be biphasic~~

~~4 (Fig. 4.9) when plotted according to the first-order rate equation:~~

~~In (B/BQ) = - k_^.t Equation 4.2~~

~~where BQ and B represent the amount of specifically bound toxin at~~

~~time zero and time t, respectively. A majority of the sites had a Fig. 4.9 Dissociation of toxin from synaptosomes prelabelled with~~

~~125I-Bo NT (A).~~

~~Rates of toxin dissociation from synaptosomes were studied at 4°C by dilution [in the absence (O) or presence (•) of excess native type A~~

~~BoNT ] of suspensions prelabelled (40 min at 37°C) with 4nM 125I-BoNT.~~

~~Following dilution (20-fold) of suspensions (at 4°C), aliquots were removed at various times, centrifuged and the pellets washed once~~

(within 6-8 min) in ice-cold buffer. Semi-logarithmic, first-order plots of specific binding are not shown to be monophasic; a dis sociation rate constant (k_^) of 5.0 x 10"^s7* was approximated for the slower dissociating component. Bq and B represent the amount of bound toxin at time zero and time t, respectively. significantly slower dissociation rate (approximately 10-fold) than the

~~remainder; an approximation to the dissociation rate constant (k ^), -5 -1 for this population of sites, was calculated as 5.0 x 10 s (in~~

~~the preence of BoNT). No significant differences were observed in k ^~~

~~for the slower dissociating component when monitored after dilution in~~

~~the presence or absence of excess native BoNT. However, under these~~

~~conditions there was a slight increase in the proportion of sites~~

~~exhibiting a faster dissociation rate, although a biphasic dissociation was still apparent; this suggests that negative cooperativity cannot~~

~~account totally for the complex binding data described. From the k+^~~

~~and derived from these kinetic experiments, the dissociation constant (Kg) is found to be 1.5nM, which is of the same order as the~~

~~Kq for the high affinity component obtained from equilibrium binding~~

~~studies. As neither the association or dissociation rates were affected 2+ significantly by the absence of Ca , or the presence of protease~~

~~inhibitors (EGTA, EDTA, phenyl methyl sulphonyl fluoride, pepstatin and~~

~~benzamidine) during the synaptosome preparation, proteolysis of the binding components ought not to be responsible for the lack of mono-~~

~~phasic kinetics. Similar results were observed with membranes of lysed~~

~~synaptosomes indicating that the binding sites are located in the membrane fraction; moreover, this finding excludes toxin internalis~~

~~ation as a contributory factor to the complicated binding data.~~

~~However, phasing of the toxin into the membrane bilayer (i.e.~~

~~sequestration) may account, at least partially, for the heterogeneity in~~

~~binding (see Discussion). The apparent complexity of the association~~

~~and dissociation kinetics (Figs. 4.8 and 4.9) is consistent with the~~

~~non-linearity of the Scatchard plot of the equilibrium binding measure~~

~~ments (Fig. 4.7). Both these results could arise from the presence of~~

~~binding sites of high and low affinity or heterogeneity in labelled~~

~~toxin species. - 151 -~~

~~Fig. 4.10 Effect of temperature and pH on synaptosomal binding of type~~

~~A 125I-Bo NT.~~

Synaptosomal suspensions (50-100ug protein) were incubated for 40 ■0. min at a series of temperature (A) or pH values at 37°C (B) with 125 I-BoNT, 2 or InM toxin, respectively. In both studies specific

~~binding ( • ) was calculated from total (o) and non-specific (■) binding~~

~~4. as detailed elsewhere. (A) Similar curves for specific binding were~~

~~obtained for a variety of 125I-BoNT concentrations examined (0.5-5nM)~~

~~(B) Krebs- phosphate buffer containing albumin was adjusted to the~~

appropriate pH using hydrochloric acid or sodium hydroxide. *

~~♦ ♦ * * * ¥~~

~~1(r4 125j_g0^j bound (dpm) 1~~

~~o hO LTI o Jl O cn o O t - "T“ “T “1~~

~~o “ eprtr (#C)temperature~~

~~Cn N>~~

~~o "~~

~~•f" O *“ 4.3.4 Effect of temperature and pH on synaptosomal binding of type A 125 125 I-BoNT. I-BoNT binding to synaptosomes was studied at a~~

~~variety of temperatures (4-37°C) at different toxin concentrations (0.5- 125 5nM). A representative figure is given, using 2nM I-BoNT, which~~

~~shows an increase in specific toxin binding with temperature between 4-~~

~~23°C; a temperature-dependent sequestration of toxin may account for~~

~~part of this increase in binding (see Discussion). At higher~~

~~temperatures (e.g. 25-37°C) a decrease in specific binding was observed~~

~~(Fig. 4.10A). This decrease is owed to a greater proportion of non~~

~~specific binding found at 37°C, compared with lower temperatures; at high temperatures (e.g. 95°C) this amount of non-specific binding is not~~

~~significantly altered (see Chapter 5).~~

~~Using buffers and toxin solutions adjusted to the required pH, the 125 interaction of I-BoNT with synaptosomes was examined (Fig. 4.10B).~~

~~Non-specific binding was not significantly altered by varying the pH;~~

~~however, a maximum value for specific binding was obtained between pH~~

~~5.5-7.5. Minimal specific binding was observed at low (pH 2.3) and high~~

~~(pH 10) pH; the latter observation is probably owed to denaturation of the toxin, since it is known to be unstable at such high pH.~~

4.3.5 Location of acceptors) for type A I-BoNT on synaptic membranes. Preliminary data suggesting that synaptosomal acceptors for 125 I-BoNT are located on synaptic membranes has already been mentioned above in that lysed membranes bound toxin to a similar extent as intact

~~synaptosomes. Further evidence for this was obtained when specific binding of I-BoNT to synaptosomal membrane fractions was examined~~

(Table 4.2). The majority of acceptors were located in the light and heavy membrane fractions which showed an enrichment of sites over the original intact synaptosomes, 155 fmol/mg protein as compared to 119

~~fmol/mg protein. The decreased content of acceptors in the myelin and - 154 -~~

~~1 Table 4.2 Binding of I-BoNT (A) to synaptosomal membraneous fractions.~~

~~Sucrose Specific binding Fraction (M) (fmol/mg protein)~~

~~Intact synaptosomes - 119~~

~~Cytoplasmic protein 0.2 N.0.~~

~~Myeli n 0.2 - 0.4 69~~

~~Synaptic vesicles 0.4 72~~

~~Light Membranes 0.4 - 0.6 135~~

~~Heavy membranes 0.6 7 1.2 155~~

~~Mitochondria 1.2 (pellet) 47~~

Purified cerebrocortical synaptosomes were lysed in 50mM Tris-HCl buffer, pH 8.0 for 30 min at 4°C and submitted to discontinuous density gradient centrifugation using 0.2, 0.4, 0.6 and 1.2M sucrose (cf. de

~~Belleroche and Bradford, 1977). Specific binding of 2nM ^ I - B o M T to the separated fractions was determined after 40 min incubation at 22°C.~~

~~(N.D. = not determined) - 155 -~~

~~Fig. 4,11 Electron-microscope autoradiographic localisation of the 125 I-BoNT (A) binding component on synaptosomes.~~

~~Synaptosomes prepared by Ficoll density-gradient centrifugation~~

~~were labelled (40 min at 37°C) with InM 125I-BoNT in the absence (A)~~

~~or presence (B) of 10~7M native BoNT. After termination of binding by~~

~~dilution in ice-cold buffer and centrifugation, the pellets were washed~~

~~twice and then fixed in buffer containing glutaraldehyde. Pellets were~~

~~then processed for electron-microscope autoradiography. Magnifications~~

~~used are as stated.~~

♦ *

~~♦ (A) 7,670 X 156~~

~~Fig. 4.11~~

~~(A) 22,670 X~~

~~(B) 7,670 X - 157 -~~

~~synaptic vesicle fraction (%70fmol/mg protein) is probably due to~~

~~cross-contamination of the fractions by light synaptic membrane~~

~~fragments. Similarly specific toxin binding to the mitochondrial pellet~~

~~is probably afforded by sedimentation of synaptosomal aggregates un~~

* dispersed by the lysis and homogenisation procedures used prior to

~~centrifugation. 125 In addition to the above, I-BoNT binding to synaptosomal~~

~~membranes has been demonstrated by electron-microscope autoradiography ♦ (Fig. 4.11). Taking into account the limitations of resolution of this~~

~~technique, the silver grains appear to be located on the synaptic~~

~~membrane (Fig. 4.11A); in control preparations very few silver grains~~

~~♦ were observed in the numerous sections examined (Fig. 4.11B). It is~~

~~interesting to note that very little, if any, labelled toxin appears to~~

~~be internalised within the synaptosomes.~~

~~% 125 4.3.6 Characterisation of the acceptor sites for type B I-BoNT on~~

~~synaptosomal membranes. In order to facilitate comparable studies on~~

~~the specific neuronal acceptors) for type B botulinum neurotoxin, the~~

~~latter was radioiodinated using identical conditions to those previously 125 developed for type A BoNT. Type B I-BoMT was found to be of high~~

~~specific radioactivity (800-1000 Ci/mmol) and retained the majority of ♦ its original toxicity (e.g. 60%). Autoradiography of labelled toxin~~

~~submitted to SDS-PAGE, in the presence of 2- mercaptoethanol (Fig. 4.12)~~

~~shows that single and dichain forms of the toxin are present. Moreover,~~

* unlike the distribution of radioactivity between the two subunits for

~~type A *^I-BoNT, with type B ^®I-BoNT only the larger of the 125 subunits showed significant incorporation of I-iodine. This~~

~~observation may be of great importance in interpretation of binding and~~

~~localisation data obtained with these labelled toxins. 158 -~~

~~Fig. 4.12 SDS-PAGE of type B 125I-BoNT.~~

♦

~~The distribution of radioactivity between the subunits of the~~

~~radio!abelled neurotoxin was examined by SDS-gel electrophoresis on a~~

~~10% polyacrylamide gel ran under reducing conditions. Mobilities of the~~

~~labelled toxin and its subunits were compared to that of native type B~~

~~BoNT and standard marker proteins by staining the gel with Coomassie~~

~~blue R-250 prior to its drying down and autoradiography. Arrows~~

~~indicate positions of stained protein bands of native type B BoNT and~~

~~its subunits. - 159 -~~

~~Fig. 4.13 Equilibrium binding of ^ I - B o N T (B) to cerebrocortical~~

~~synaptosomes.~~

~~(A) Synaptosomes were incubated (60 min at 37°C) with increasing~~

~~concentrations of type B 125I-BoNT (0.1-17.5nM). Binding was~~

~~terminated and specific binding (•) was determined by subtraction of~~

~~non-specific (■) from total (O) binding (described in Fig. 4.7).~~

~~(B) A Scatchard plot for the specific binding was biphasic, represent~~

~~able of high-affinity (KQ = 0.5nM, Bmax = 30fmol/mg protein) and~~

~~low-affinity (Kg ^20nM, Bmax = 3pmol/mg protein); components, as~~

~~illustrated by graphical analysis.~~

~~♦ cr» O~~

~~Fig. 4.13 ♦ I-BoNT bound bound I-BoNT (pmol/mg protein) ♦ # 125 9 oo NJ Ln I-BoNT(B) InM] 9 j K 9 j\ 9 -i* S J o~~

~~0*25 0-5 Bound toxin (pmol/mg protein) - 161 -~~

~~125 Fig. 4.14 Association of type B I-BoNT with acceptors on~~

~~synaptosomal membranes.~~

~~Synaptosome suspensions (50-100pg/mg protein) were incubated with~~

~~InM I-BoNT at 4°C for various time intervals after which aliquots~~

~~were taken, diluted in ice-cold buffer, centrifuged and washed once~~

~~before quantitation of bound toxin. (A) Specific binding (•) was~~

~~calculated from total (o) and non-specific (■) binding as detailed~~

~~earlier. Error bars represent the standard error of mean values (n=3).~~

~~(B) A second order rate plot (see Section 4.3.3, equation 4.1) of~~

~~specific binding calculated in (A) is curvilinear between 0-15min. An~~

~~association rate constant (k+1) of 4.5 x 10^ M.“*s“* was~~

~~approximated from the initial rate of reaction (0-5min).~~

~~♦ - 162 -~~

~~Fig. 4.14~~

♦

* Fig. 4.15 Dissociation of toxin from synaptosomes prelabelled with

~~125I-Bo NT (B).~~

~~1 Synaptosomes were prelabelled with InM I-BoNT for 40 min at~~

37°C prior to cooling on ice to 4°C. Dissociation was initiated by dilution of suspensions (25-fold) with ice-cold buffer containing albumin, in the absence (o) or presence (•) of excess native toxin.

Aliquots were taken at various time intervals (0-180 min), centrifuged and washed once in the above buffer before submitting to Y-radiation counting. Specific binding presented on a first-order semilogarithmic plot was not mono-phasic; BQ and B represent the specific toxin binding at time zero and time t respectively. An approximation of the dissociation rate constant (k ^) of 2 .1 x 10“^s.~* was made for the slower dissociating component. 195 Saturable binding of type B I-BoNT to rat cerebrocortical

~~synaptosomes was also demonstrated (Fig. 4.13); a Scatchard plot (Fig.~~

4.13B) of the data, obtained from the concentration dependence of toxin binding (Fig. 4.13A), revealed a heterogeneous population of sites 125 similar to those observed for type A I-BoNT (Fig. 4.7). Graphical analysis of the curve (Fig. 4.13B) showed a low content of high affinity sites (Kg = 0.5nM, Bmax = 30 fmol/mg) and a much larger content of low affinity sites (Kg %20nM, Bmax x 3pmol/mg protein). As already 125 shown for type A I-BoNT, lysis of synaptosomes did not 125 significantly affect the observed level of binding for type B I-

BoNT. The similarity in shape of the Scatchard plots for equilibrium 125 binding of types A and B I-BoNT tends to suggest that the complexities observed are more likely to be due to heterogeneity of acceptor populations than of the labelled toxins; this view is strengthened by the observation that the distribution of radiolabel in these two toxins is very different. 125 Kinetics of the interaction of type B I-BoMT with its synaptosomal acceptors were studied at 4-C using InM labelled neurotoxin in order to study mainly the high-affinity acceptors. Heterogeneity in association (Fig. 4.14) and dissociation (Fig. 4.15) rates was similar 125 to that observed above with type A I-BoNT. A second order plot

(Fig. 4.14B) of the specific binding (obtained from Fig. 4.14A) was biphasic; from the initial rate of binding (i.e. for the faster- associating component), k+^ = 4.5 x 10^ M“*.s“* was approximated. A semi-logarithmic plot for dissociation of specifically bound toxin from prelabelled synaptosomes (see equation 4.2, Section

~~4.3.3) was also biphasic (Fig. 4.15); an increased initial rate of dis~~

~~sociation was apparent when carried out in the presence of unlabelled~~

~~BoNT. However, not all the sites exhibited rapid dissociation in the~~

presence of excess native toxin, which suggests that even if negative cooperativity in toxin binding exists, it could only partially explain the heterogeneous dissociation rates observed. An approximation of k_^ = 2 .1 x 1 0 -5 s“*. was calculated for the slower dissociating component in the presence of BoNT. Using these values obtained for k+1 and k ^ , the calculated KQ was 0.5nM; this value is the same as that obtained for equilibrium binding measurements for the higher affinity site (Fig. 4.13B).

~~4.4 DISCUSSION~~

Significant progress towards the understanding of the molecular mechanism(s) of transmitter release and the unique potency of botulinum neurotoxins should be achievable with the availability of a biologically active, radiolabelled derivative of these homogeneous proteins. Such 125 I-iodinated preparations of high specific radioactivity and stability, are reported here for the first time; they have facilitated notable advances in the characterisation of their binding sites(s) in the central nervous system (described herein), and at the mammalian neuromuscular junction (Dolly et al_., 1982, 1984a; Black et al_., 1983). 125 Previous preparations of I-labelled toxin complex or neuro toxin were either of low specific radioactivity (Habermann, 1974; Kozaki and Sakaguchi, 1982) or unstable upon storage (Kitamura, 1976; Kozaki,

1979). Also, when neurotoxin was purified in our laboratory, by the sole use of ion-exchange chromatography (as employed in some of the latter studies), it contained variable amounts of a contaminating protein, Mr of 130000 (Tse et al_., 1982), exhibiting little or no toxicity; such a extraneous protein could become radiolabelled and 125 125 complicate investigations on I-BoNT. In the case of the I-

BoNT described in this study, homogeneous neurotoxin (see Chapter 2) was used in its preparation; also, its high degree of radio!abelling (700- 1750 Ci/mmol) was accurately determined and its neurotoxicity shown to be almost unchanged (60-85% of original) from native neurotoxin.

Apparently, the residues iodinated are inessential for its toxicity; however, radioiodination may result in a decreased binding affinity of the toxin for synaptosomes. Lack of detectable changes in the immuno- 1 o c reactivity of type A A^I-BoNT suggests no gross structural alteration on radiolabelling. The observed neurotoxicity could only be attributed partially to the presence of unlabelled toxin, since in many cases the specific radioactivity of the preparations approached 2000 Ci/mmol, 125 which would give an equimolar ratio of I-iodine to protein. In 125 view of this one might expect the majority of I-BoNT to be represented by mono-labelled species with only a negligible fraction of doubly or higher labelled species present. If this is the case, some inherent microheterogeneity must exist in the mono-iodinated toxin since it has been shown that either of the two subunits of type A may be labelled (Fig. 4.2). Several methods employed for resolving iodinated species and unlabelled neurotoxin (e.g. narrow range IEF, chromato focussing and ion- exchange chromatography) have so far been unsuccessful. However, there is some evidence from the latter method that native neurotoxin may elute prior to the iodinated species, although resolution of peaks would be very poor. Conclusive evidence 125 for the biological activity of type A I-BoNT , at least in terms of specific interaction with nerve membranes, has been illustrated by the autoradiographic localisation in the light microscope of saturable sites at motor nerve terminals (Dolly et al_., 1981, 1982). This could not be achieved using preparations that had lost a large proportion of their neurotoxicity; also, such samples exhibited unacceptably high degrees of non-specific binding to brain synaptic membranes.

The presence of saturable binding sites for I-BoNT (A and B) on brain synaptosomes has been demonstrated by equilibrium and kinetic methods. Partial resolution of the Scatchard plots has been achieved by

~~graphical analysis (Rosenthal, 1967), where calculations show high-~~

~~affinity components with and content of sites compatible with those~~

~~expected for a potent neurotoxin, suggesting that these may be~~

~~functionally significant components. Characteristics of the toxin~~

~~binding sites were further examined by kinetic analysis; in these~~

~~studies relatively low toxin concentrations were used in order to~~

~~observe primarily the high-affinity acceptor(s). However, under such~~

~~conditions the kinetics of association and dissociation were complex; in~~

~~addition, it was apparent that the proportion of sites exhibiting fast~~

~~association and slow dissociation rates did not correspond directly to~~

~~the population of sites showing high-affinity for the toxin in~~

~~equilibrium binding measurements. The observed heterogeneity in the 125 high-affinity binding of types A and B I-BoNT to synaptosomes may~~

~~be due to the inherent microheterogeneity of the labelled toxin species~~

~~(see below), together with a contribution by the large number of~~

~~low-affinity acceptors present. This population of low-affinity sites~~

~~is not thought to be due to a decreased affinity of the toxin for its~~

~~acceptor(s), as a result of radioiodination, since their content is so~~

~~large ( > 2pmol/mg protein); however, it may be due to^saturable~~

~~population of 'non-specific' sites. Owing to the aforementioned~~

~~complications of the kinetic data, only approximations could be made of~~

~~the association and dissociation rate constants. Using the estimates of~~

~~these values, the values derived were 1.5nM and 0.5nM for types A 125 and B I-BoNT, respectively; these are close to the values obtained from equilibrium binding discussed above. These high- affinity~~

~~sites could not be distinguished in competition experiments (see Chapter~~

~~5, Fig. 5.6), possibly due to their being masked by the much higher content of low-affinity sites, as apparent in the Scatchard plots~~

(Figs. 4.7, 4.13). Using the values obtained from the concentration dependence of binding, a semi-logarithmic graph in which type A 125 I-BoNT bound is plotted against the concentration of free toxin, on

~~a logarithmic scale, was not sigmoidal due to some sites being~~

~~unoccupied. As the affinity of the latter is unknown, it is not possible to determine accurately the concentration or of these lower-affinity sites. Recently, synaptosomal binding sites for~~

~~iodinated botulinum neurotoxins (types A, B and E) have been reported in brief (Kozaki, 1979; Kozaki and Sakaguchi, 1982) but it would appear~~

~~that they exhibited very low affinity for the radiolabelled toxin used~~

~~(at least with type E toxin, KQ -vlOOnM) which itself was of low specific radioactivity ( < lOOCi/mmol).~~

Although relatively low concentrations of toxin were used for kinetic studies on predominantly the high-affinity component, association and dissociation rates were still found to be biphasic. The binding data was unchanged when protease inhibitors were included in the medium (observed for type A toxin) or lysed synaptosomes were used

(shown for types A and B toxin). A lack of monophasic kinetics, and the presence of heterogeneity in equilibrium measurements should not, therefore, be attributed to proteolysis of a single binding component or complications arising from any internalisation of toxin within synaptosomes; of course, phasing of toxin into the membrane cannot be excluded as proposed for tetanus toxin (Yavin et al_., 1983). Although a small increase in the number of faster-dissociating components was apparent when dissociation was performed by dilution in the presence of an excess of BoNT, the kinetics were not monophasic. This suggests that there may be some cooperativity in the binding of types A and 3 125 I-BoNT but this cannot account exclusively for the biphasic kinetics observed. Owing to the distribution of radiolabel between both of the toxin's subunits (at least with type A)and the inability to separate the different labelled species, one cannot exclude the unavoidable presence of microheterogeneity in the toxin which could contribute to the complexity of both the kinetic and equilibrium studies.

The binding of type A I-BoMT to its synaptosomal acceptor(s) appeared temperature-sensitive between 4-20°C. Simpson (1980), using indirect electrophysiological methods and nerve muscle preparations, reported that binding was not particularly sensitive to temperature, having a low Q^q K l . 6). This does not conflict with the above data from synaptosomes as Simpson (1980) only investigated the effect of temperature on binding between 25-35°C. As seen in Fig. 4.10A, there is little difference in the ability of synaptosomes to bind labelled toxin in this temperature range. Using similar electrophysiological methods,

Schmitt et a K (1981) found no difference in the binding of tetanus toxin between 18-37°C. However, when direct binding studies were 125 performed on neuronal cell cultures using I-labelled toxin an increase in binding was observed at 0°C relative to that at 28°C (Yavin et al_., 1981). These authors suggest that the lower level of binding at

28®C may reflect interactions of toxin with acceptors that exhibit higher affinity at low temperatures than at 28°C. The physiological role of acceptors that preferentially binds toxin at non-physiological temperatures (0-4°C) remains to be explained. The decrease in type A 125 I-BoNT binding to synaptosomes at 4 C is similar to that seen at the NMJ, studied under similar conditions (Dolly et al_., 1984a), although in this latter study it is not certain how much diffusion problems contribute to this. 125 The binding of I-BoNT to synaptosomes showed a very broad pH optimum, suggesting that specific ionisations are not of paramount importance in these toxin-acceptor interactions. However, it is known that sialic acid residues are involved in toxin binding (see Chapter 5;

Williams et a K , 1983); in which case the removal of their negative charges in acidic conditions may contribute, at least partially, to the decreased binding observed at low pH values. The progressive decrease in ability of toxin to bind synaptosomes at pH values above 7.5 is most* probably a reflection of the instability of botulinum neurotoxin in alkaline conditions. 125 It is noteworthy that the binding sites for I-BoNT were located on synaptosomal membranes; following fractionation of synaptosomes prelabelled with type A I-BoNT, the bulk (*u70%) of the radioactivity was found to be associated with the synaptic membrane fractions (Dolly et a K , 1982). Preferential binding of *^I-BoNT (A) to synaptosomal membranes was also shown by incubation of membrane components with labelled toxin subsequent to their fractionation. 125 Interaction of type A I-BoNT with other fractions probably results from cross-contamination by membranes between fractions on the sucrose gradient and incomplete lysis of synaptosomes prior to fractionation; however, it is possible that some of this binding is due to interaction of toxin with an intracellular site(s), although at present no sub stantial evidence for this exists. Localisation of specific synaptosomal acceptors for botulinum neurotoxin has now been achieved by electron-microscope autoradiography of synaptosomes prelabelled with 125 type A I-BoNT. It is very noticable that in such studies minimal internalisation of bound labelled toxin occurs (Fig. 4.11A); this is in great contrast to the distribution of silver grains at neuromuscular 125 junction in preparations similarly incubated with type A I-BoNT

(Dolly et al_., 1984a), where about 40% of the silver grains are located within the nerve terminal. However, it is well documented that synaptosomes retain many of the normal functional properties of intact neurons (cf. Fried and Blaustein, 1976); in addition, horseradish peroxidase (Mr -v.40000) has been shown to be taken up by synaptosomes

~~(Marchbanks, 1982) and at the frog neuromuscular junction (Litchy, - 171 -~~

~~1973). Thus, it is likely that the lack of internalisation of 125 I-BoNT into synaptosomes is a selective phenomenon and not due to a~~

~~general malfunction of uptake mechanisms. This demonstrated inability~~

~~of synaptosomes to internalise BoNT may explain the apparent non-toxic~~

~~♦ nature of this protein centrally, following intraventricular injection~~

~~into rat brains (Williams et al_., 1983).~~

~~The advent of biologically active ^I-lab el le d goMT (types A~~

~~and B) of high specific radioactivity has allowed their saturable ♦ acceptors) on neuronal membranes in the central nervous system~~

~~(described above) and the periphery (Black et a K , 1983; Dolly et al.,~~

~~1984a) to be characterised. Having demonstrated the specific binding of * 125 types A and B I-BoNT to acceptors in the CNS, studies into the~~

~~nature of such specific interactions are now made possible.~~

♦

~~ft - 172 -~~

♦

~~CHAPTER 5~~

♦

~~NATURE AND SELECTIVITY OF NEURONAL RECEPTORS FOR BONT~~

♦

* 5.1 INTRODUCTION

~~Most, if not all, external components of neuronal membranes are~~

~~glycosylated (Hakomori, 1975) and hence the distribution, structure and~~

~~activity of membrane glycoproteins and glycolipids are of great general~~

~~interest as candidates for mediating intra- and inter-membraneous inter~~

~~actions. Integral membrane proteins interact with both hydrophilic and hydrophobic regions of the membrane and exhibit strong interactions with~~

~~adjacent lipids (Singer and Nicolson, 1972). Such macromolecules as~~

~~receptors would be able to offer specificity in their interaction~~

~~through their hydrophilic glycosylated region; if the ligand acts at an~~

~~intracellular site then its insertion through the membrane may be~~

~~assisted by the receptor's hydrophobic domains.~~

~~Also, glycolipids (i.e. gangliosides) being primarily membrane components, having hydrophilic and hydrophobic regions (Nicolson and~~

Singer, 1974; Fishman and Brady, 1976), may act as versatile receptor molecules. The interaction of these small mobile molecules with external ligands may show great specificity since the composition of the

~~externally located glycosylated portion may vary greatly. In relation to BoNT binding to specific membrane components of the nervous system,~~

~~it is interesting that neural tissue is extremely rich in the higher-~~

~~sialated gangliosides (Wiegandt, 1972).However, early studies failed~~

~~to find any interaction of botulinum toxins with gangliosides (van~~

~~Heyningen and Mellanby, 1973; Mellanby et al^., 1973; Habermann, 1974;~~

~~Habermann and Heller, 1975). More recently, a detoxification of type A~~

~~BoNT by gangliosides has been reported (Kitamura et al_., 1980) using~~

~~indirect methods; the number and position of sialic acid residues is~~

~~suggested to be important in these interactions, the most effective of~~

~~the gangliosides being the trisialoganglioside GTlt). Their observation that inactive *“ I-labelled toxin complexes with the ganglioside questions the specificity of these findings. However, the~~

~~specific toxin-gang!ioside interaction these authors report may be mediated by the toxin's L-subunit which may remain intact after~~

inactivation; the decrease in toxicity on inactivation being due to an effect on the H-subunit. Moreover, Wonnacott (1980) found no shift in the dose response curve for inhibition of acetylcholine release by BoNT when gangliosides were preincubated with brain synaptosomes. Tetanus toxin, as discussed previously (see General Introduction), has a very similar subunit structure and pharmacological action to BoNT. Treatment of primary culture cells (Dimpfel and Habermann, 1977), neuroblastoma cells (Zimmerman and Piffaretti, 1977) or synaptosomes (Habermann et al., 1981) with neuraminidase greatly reduces the binding of tetanus toxin to these preparations, as would be expected if the gangliosides

G D ^ and GT^ are involved as proposed (Dimpfel et al_., 1977; Ledley et al^., 1977). To date, only one group has studied the effect of neuraminidase on the binding components for botulinum toxin; they showed a decrease in toxin binding to rat brain synaptosomes (Habermann and

~~Heller, 1975); this suggests sialic acid (N-acetylneuraminic acid,~~

NANA) residues may be involved, although these workers failed to show any effect of gangliosides on binding. Also, neuraminidase treatment of brain preparations did not appear to affect the action of BoNT on transmitter uptake or release (Bigalke et al_., 1981; Habermann et al.,

~~1981).~~

Lectins are proteins which interact with specific mono- and oligo saccharides. Many lectins are readily available in pure forms and their binding can be inhibited by simple sugars; hence they have been widely used to characterise the distribution (Nicolson, 1974; Sharon and Lis,

~~1975; Kelly et a1_., 1976) and structure (Sharon and Lis, 1972; Nicolson,~~

1974; Capeau and Picard, 1980) of cell membrane saccharide-containing components. Therefore, with the use of such lectins it may be possible to confirm, and reveal additional, specific interactions of BoNT with sugar or sugar-containing moieties, thereby providing further evidence as to whether the acceptors involved are glycoproteins and/or glycolipids.

To study membraneous receptors in more detail most procedures require their partial or complete purification in an active solubilised form. However, a technique used more and more frequently nowadays is irradiation inactivation; this allows estimation of the functional size of membrane-bound molecules without their purification (reviewed by

Kempner and Schlegel, 1977). This method utilises the fact that biologically active molecules progressively lose their activity on increasing exposure to high-energy radiation. Since the measurements made after irradiation are biological activities, this technique, unlike others available, describes a functional unit rather than a structural size. The usefulness of this technique is well illustrated by recent reports of oligomeric sizes for the nicotinic acetylcholine receptor (Lo et al_., 1982), benzodiazepine receptor (Chang and Barnard, 1982; Doble and Iversen, 1982; Ferry et al_., 1983; Paul et a1_., 1981), calcium channels (Ferry et al_., 1983; Venter et al_., 1983) and opiate receptors

~~(Ott et a K , 1983). It is hoped that this methodology may be applicable to the saturable acceptors for BoNT.~~

Many bacterial toxins (e.g. cholera, tetanus, and diphtheria toxins; see General Introduction) and some glycoprotein hormones (e.g. thyrotropin, luteinising hormone and human chorionic gonadotropin; see van Heyningen, 1982b) are shown to have two or more heterologous subunits. Of these subunits, one type is thought to specifically inter act with cellular receptors whilst the other exerts the molecule's physiological effect, although such action may not be the direct result of the initial receptor binding. The most characterised of such inter actions is that of cholera toxin which specifically binds to the monosialoganglioside GM^ through its type 3-subunits (van Heyningen,

~~1977). These subunits have no other known function and it is the A- subunit which is essential in activating the enzyme adenylate cyclase.~~

It is interesting to note that tetanus toxin is also thought to bind its ganglioside acceptors by its larger subunit (Matsuda and Yoneda, 1975; van Heyningen, 1976). As found with tetanus toxin (Matsuda and Yoneda,

~~1975), antigenic differences are reported in the separated subunits of type 8 BoNT (Kozaki and Sakaguchi, 1975) and type A BoNT (see Chapter 2)~~

~~Whether such binding of 3oNT to its acceptors is mediated by one or other of its subunits remains to be established. A previous report~~

(Kozaki, 1979) using type B BoMT suggests that binding may be via the larger subunit; however, binding of a label!ed-neurotoxin derivative to synaptosome suspensions was significantly inhibited by the smaller polypeptide. Moreover, it is not yet known whether each of the eight imrnunologically distinct botulinum neurotoxins have the same or different acceptors and if they exhibit the same binding affinity.

In the following study the nature of the specific synaptosomal acceptors for BoNT (A and B) are examined together with the effect of high-energy radiation on these components in an attempt to define their functional size. In addition, the selectivity of such specific toxin acceptors on synaptosomal membranes is examined by the interaction of several other presynaptically active neurotoxins and botulism antagonists thereon.

~~5.2 METHODS~~

~~5.2.1 Investigations into the nature of acceptors for types A and B~~

I-BoNT. Types A and B I-BoMT and synaptosome preparations used in these studies were prepared as in Chapter 4. All incubations of membranes described in this section (except protease digestions) were carried out in Krebs-phosphate buffer, pH 7.4, containing albumin

~~(lmg/ml).~~

~~The possible proteinaceous nature of the binding component(s) was studied by preincubation of membranes with proteinase K (protease from~~

Tritichariurn album) or TPCK-trypsin (both proteases at 0.1-lmg/ml final concentration, 0-30 min at 37°C) or by heating (0-20 min at 95°C) prior to incubation with A^3I-BoNT. Digestion with proteinase K or trypsin

(with specific activities of 13 units and 12300 BAEE units per mg protein, respectively) was terminated by the addition of buffer containing PMSF or trypsin inhibitor respectively; both inhibitors were added to a 5-fold excess over that required for the inhibition of the amount of protease present. Membranes were then centrifuged, washed with buffer (containing inhibitor) and resuspended in the same buffer 125 containing albumin before incubation with type A or B I-BoMT.

~~Control membrane samples were preincubated with proteinase K or trypsin in the presence of excess inhibitor.~~

~~The effect of the following non-proteolytic enzymic digestions~~

(0.5 units/ml, 40 min at 37°C; unless otherwise stated in figure legends) on the toxin binding capacity of synaptosomal membranes was investigated; enzymes used were neuraminidases from Clostridium perfringens and Arthrobacter ureafaciens, N-acetyl-0-glucosaminidase, a-

L-fucosidase and 0-galactosidase. The specificity of neuraminidase action was confirmed by preincubation (20 min at 37°C) of the enzyme with 20mM 2, 3-dehydro-2-deoxy-NANA (from Sigma), a specific inhibitor of neuraminidase. Following enzymic digestion, membranes were washed twice prior to incubation with radiolabelled toxins. p c The influence of a variety of lectins on I-BoNT (A and B) binding to synaptosomes was examined. These included concanavalin A

~~(Con A) from Canavalia ensiformis, soyabean agglutinin (SBA) from~~

~~Glycine max, Ricinus communis agglutinin type I (RCAj) from castor beans and wheat germ agglutinin (WGA) from Triticum vulgaris. The saccharide-binding specificities of these lectins were (Lotan and~~

Nicolson, 1979; Capeau and Picard, 1980): Con A, terminal or internal o-mannose and a-glucose; SBA, terminal galactose and M-acetyl-galacto- samine; RCAj, terminal 3-galactose; WGA, terminal or internal

~~N-acetyl-glucosamine (NAG) and N-acetyl-neuraminic acid. Preincubation of synaptosomes with lectins (60 min) was performed at either 4~C (SBA,~~

~~RCA, WGA) or at 22°C (Con A) to ensure optimal conditions for binding~~

(Lotan et a K , 1977), after which membranes were washed with ice-cold 125 buffer. Binding of type A or 3 I-BoNT was subsequently performed at 4°C. Toxin binding to treated membranes (with non-proteolytic enzymes or lectins was initiated by addition of I-BoNT; the concentrations of toxin used are detailed in figure legends. Binding was terminated after 40 min at 37°C (unless stated otherwise) by dilution and centrifugation; specific binding was calculated from total and non-specific binding as detailed previously.

Gangliosides (1 mg/ml) or sialic acid (3mM) were incubated with synaptosome suspensions for 40 min at 37°C; after washing in buffer, 125 membranes were resuspended in buffer containing type A I-BoNT in 125 the presence or absence of the latter compounds. I-BoMT binding to membranes was terminated after 40 min at 37°C and specific binding was 125 determined as detailed above. Otherwise, 1.3nM type B I-BoNT was incubated with gangliosides (0-1.0 mg/ml, 40 min at 37°C) before initiation of binding by the addition of synaptosome suspension.

~~Neuraminidase (from Arthrobacter ureafaciens) was supplied by~~

~~Calbiochem-Behring Corporation. Other enzymes, lectins and gangliosides~~

~~(type IV) used in this study were obtained from Sigma. A unit of neuraminidase will liberate lurnol of N-acetylneuraminic acid/min at pH~~

~~5.0 at 37°C using N-acetyl-neuraminyl-lactose as substrate; one unit of proteinase k will hydrolyse casein to produce colour equivalent to lumol - 179 -~~

~~of tyrosine/min at pH 7.5 at 37°C (colour by Folin-Ciocalteu reagent);~~

~~one 3AEE unit = A ^2$-$ of 0.001/min, with Ma-benzoyl-L^arginine ethyl~~

~~ester (BAEE) as substrate in 3.2 ml at pH 7.6 at 25°C: N-acetyl-8-~~

~~glucosaminidase, one unit will hydrolyse lumol of p-nitrophenyl-N-~~

~~♦ acetyl-3-D-glucosaminide to p-nitrophenol and N- acetyl-D-glucosamine/~~

~~min at 25°C; a-L-fucosidase, one unit hydrolyses ljimol of p-nitrophenyl-~~

~~a-L-fucosidase to p-nitrophenol and L-fucose/min at pH 6.5 at 25°C;~~

~~3-galactosidase, one unit will hydrolyse lpmol of o-nitrophenyl-S-D- *• galactoside to o-nitrophenol/min at pH 7.3 at 37°C.~~

~~5.2.2 High-energy radiation of synaptosomal membranes and standard~~

* enzyme markers. The general principle underlying this technique is

~~based on the observation that high energy radiation, when passing~~

~~through biological matter, transfers energy to the irradiated material~~

~~in the form of randomly distributed primary ionisations. When occuring~~

~~in a biologically-active molecule, they destroy its function; any~~

~~remaining activity is therefore thought to be due to molecules escaping~~

~~bombardment. Thus, the decrease in biological activity is directly * related to its functional size (Kempner and Schlegel, 1979).~~

~~Irradiation studies were performed essentially according to the~~

~~modified method of Lo et al_. (1982). Suspensions of synaptosomes (1.5~~

* mg/ml) in Krebs-phosphate buffer, pH 7.4, containing 3- galactosidase

~~(10 units/ml) were aliquoted (200pl/tube) into pyrex glass tubes and~~

~~frozen at -80°C. Other tubes containing enzyme molecular weight markers~~

~~for calibration (2 units of each enzyme/tube) were also frozen at~~

~~-80°C; enzymes used (purest grades from Sigma) were: 3-galactosidase~~

~~(from E. coTi), Mr464000; pyruvate kinase (from rabbit muscle), Mr~~

~~224000; phosphocreatine kinase (from rabbit muscle), Mr 170000 and~~

~~alcohol dehydrogenase (from horse liver), M 84000. Frozen samples~~

~~were lyophilised (^15h) and sealed under high vacuum ( < 2 x 10“^mm Hg) taking care not to heat the dried samples; sealed tubes were stored at -80°C until irradiated.~~

~~Irradiation was performed with the 3 MeV linear accelerator at~~

~~Hammersmith Hospital, London. Samples in an aluminium holder, cooled in a dry ice/acetone bath, were held in a uniform beam at a dosage of 0.4~~

Mrad/min for increasing lengths of time, to give various integrated radiation dosages. Irradiated samples were subsequently stored at -80°C until toxin binding and enzyme assays were to be performed. The contents of the tubes were then resuspended in 5mM Tris-HCl buffer, pH

~~7.5, containing bovine serum albumin (1 mg/ml).~~

~~Toxin binding to irradiated membranes was carried out using 2nM type A 125I-Bo NT or 8.3nM type B 125I-BoNT for 40 min at 37°C.~~

Binding was terminated and quantified as described previously. The enzyme assays used (described below) were those as previously reported but adapted to automated sampling and recording of activity on the LKB

~~8600 reaction rate analyser; phosphocreatine kinase and pyruvate kinase~~

~~(Liu et a K , 1980); alcohol dehydrogenase (Valle and Hoch, 1955) and 0- galactosidase (Craven et al_., 1965):~~

~~Phosphocreatine kinase:~~

~~Solution A: Q.1M triethanolamine buffer, pH 7.0, containing 20mM~~

~~glucose, lOmM magnesium acetate, ImM ADP, lOmM AMP, 0.6mM~~

~~NADP, hexokinase (1.25 units/ml), glucose 6-phosphate~~

~~dehydrogenase (1.25 units/ml) and 9mM glutathione.~~

~~Solution B: 35mM creatine phosphate. - 181 -~~

~~Pyruvate kinase:~~

~~Solution A: 50mM Tris-HCl buffer, pH 7.5, containing 0.1M KC1, 20mM~~

~~MgSO^, lmM EDTA, 0.25mM NADH, 7.5mM phosphoenolpyruvate « and lactate dehydrogenase (25 units/ml).~~

~~Solution B: 0.1M ADP.~~

~~#• Alcohol dehydrogenase~~

~~Solution A: lOOmM Tris-HCl buffer, pH 8.8, 0.2M ethanol.~~

Solution B: 50mM NAD *

~~g-galactosidase~~

~~Solution A: 50mM sodium phosphate buffer, pH 7.5, 0.01M 2-~~

~~mercaptoethanol, lmM MgClg.~~

~~Solution B: 50mM o-nitrophenylgalactopyranoside.~~

~~4. In each case, solution B (25|il) was automatically dispensed and mixed~~

~~with solution A (0.5ml) containing the enzyme sample (10-25ul); the~~

~~rate of change in absorbance at 340nm (creatine phosphokinase, pyruvate~~

~~m kinase and alcohol dehydrogenase) or 410nm (g-galactosidase) was~~

~~recorded.~~

~~125 5.2.3 Studies on the selectivity of synaptosomal acceptors for I-~~

~~BoNT (A and B). Abilities of native type A BoNT and its separated sub~~

~~units, type B BoNT, other presynaptically active toxins and antagonists~~

~~of botulism to compete with I-BoNT (A or B) for its synaptosomal~~

~~binding site(s) were studied at 37*C as follows. Type A or B I-~~

~~BoNT (l-2nM) was incubated with synaptosomes in the presence of~~

increasing concentrations (0-luM) of test compound for 40 min; binding - 132 - was terminated by dilution and centrifugation as above. Control experiments with toxins exhibiting phospholipase activity were performed in Ca~ -free buffers (and in the presence of lmM EDTA) or incubations were at 4°C.

~~Both polypeptide chains of type A BoNT were separated by QAE-~~

Sephadex anion-exchange chromatography in the presence of reducing agent and urea (see Chapter 2). Each was dialysed against 25mM sodium phosphate buffer, pH 7.5, containing 0.1M NaCl for 48hr before use in competition assays with type A AC,JI-BoNT.

The pure preparations of 3-bungarotoxin, dendrotoxin, taipoxin, a- latrotoxin and phospholipase Ag from bee venom and Naja naja melanoleuca are detailed elsewhere (Othman et a1_., 1982). Purified crotoxin (Chang and Lee, 1977) from the venom of Crotalus durissus cascavella and tetanus toxin isolated (van Heyningen, 1976) from

~~Clostridium tetani were provided by Drs. 3. Hawgood and P.0. Walker.~~

~~5.2.4 Preparation of ^ 1 - labelled subunits from BoNT. Two- dimensional-PAGE was carried out by IEF (O'Farrell, 1975) in the first dimension (in the presence of urea and reducing agent) and SDS-PAGE~~

(under reducing conditions) in the second dimension. IEF gels were prepared using a solution containing 5.5g urea (ultrapure grade), 1.33ml acrylamide (28.38% acrylamide/1.62% bis-acrylamide), 0.5ml Pharmalytes

(pH 5-8) and 3.97ml deionised water; after degassing, crosslinking of the acrylamide solution was catalysed by the addition of 10% ammonium persulphate (lOpl) and 75% TEMED (7pl). Gels (13 x 0.25cm) were poured into glass tubes, overlaid with 8M urea and allowed to set (1 hr). The urea overlay was then replaced with 20jil of lysis buffer (9.5M urea, 2%

~~Pharmalytes [pH 5-8], 5% 2-mercaptoethanol) and overlaid with a small amount of deionised water. After 1 hr these overlays were replaced with~~

~~20ul fresh lysis buffer; gels were pre-electrophoresed at 200V (15 min), - 183 -~~

~~300V (30 min) and 400V (30 min) with degassed 20mM NaOH at the cathode 125 and lQmM H^po^ at the anode. Type A I-BoMT was loaded in lysis~~

~~buffer and overlayed with 10pl of 9M urea containing 1% Pharmalytes (pH~~

~~5-8). Focussing was performed (at 400V) overnight (15 hr) at 4°C, after~~

~~which gels were sliced in half longitudinally; one half was used for~~

~~determination of the pH gradient whilst the other was equilibrated (30~~

~~min) in SDS- sample buffer containing 2-mercaptoethanol before placing~~

~~onto the stacking gel of a 10% SDS-polyacrylamide resolving gel (see * Chapter 4, Section 4.2.2). The IEF gel was sealed in place with 1%~~

~~Agarose in SDS-sample buffer containing reducing agent. ^^I-labelled~~

~~marker proteins were run in a track at the side of the SDS-gel.~~

* Following electrophoresis (see Section 4.2.2.), the gel was fixed (25%

~~isopropanol/10% acetic acid), washed thoroughly with deionised water,~~

~~dried and submitted to autoradiography.~~

The H-subunit (Mp 91000) from type A BoNT was prepared and ♦ renatured in sodium phosphate buffer as detailed earlier (Chapter 2); it 125 was radiolabelled with I-iodine according to the method described

in Chapter 4 for the iodination of intact type A BoNT. *

~~♦ - 184 -~~

~~5.3 RESULTS~~

~~125 5.3.1 Nature of saturable acceptors for types A and B I-BoNT on~~

~~synaptosomal membranes. To determine whether the binding component(s)~~

~~involved were proteins, synaptosomes were preincubated with trypsin ♦ followed by an excess of trypsin inhibitor and washing prior to the~~

~~addition of labelled toxin. Suspensions treated in such a manner failed 125 to show any specific binding when 0.2nM type A I-BoNT was used~~

~~% (Table 5.1), a concentration of toxin that should preferentially occupy~~

~~the high-affinity sites (cf. Fig. 4.7). The presence of an excess of~~

~~trypsin inhibitor in a control incubation with trypsin prevented~~

~~inactivation of the binding (Table 5.1). Susceptibility of the binding * component for type A BoNT to proteolysis was confirmed by preincubation~~

~~of synaptosomes with proteinase K, followed by inhibition of its~~

~~activity by the addition of PMSF and its removal by washing; such » treated membranes failed to exhibit any specific binding of labelled~~

~~toxin (Table 5.1). Furthermore, the binding of type A I-BoMT was~~

~~inactivated by heating; incubation of synaptosome suspensions for 2 min~~

* at 95~C removed essentially all the specific high-affinity binding 125 (Table 5.1). As expected, heat treatment of I-BoNT (A and B)

~~destroyed their abilities to bind specifically to synaptosomes (data not~~

shown). In contrast to the above observations, trypsin treatment of * membranes resulted in only a partial inhibition of specific type 3 125 I-BoNT binding using InM toxin (Fig. 5.1A). Similarly, heat 125 treatment of membranes only partially reduced specific type B I- # BoNT binding when high concentrations were used (lOnM), although no

~~inhibition was evident when low concentrations (0.5nM) were used (Fig.~~

~~5.IB). Thus it appears that populations of acceptors exist for type B~~

~~BoNT which exhibit differential sensitivities to heat and~~

~~trypsinisation; in the case of heat-resistant sites (and possibly the~~

~~trypsin-resistant ones also), these appear to be of high affinity. Table 5.1 Nature of the high-affinity synaptosomal acceptor for BoNT~~

~~(A).~~

~~Treatment Relative specific binding [%)~~

~~None (total binding) 100 ± 9~~

~~Trypsin (lmg/ml) 0~~

~~Trypsin + trypsin inhibitor 118 ± 5~~

~~Proteinase k (lmg/ml) 0~~

~~Proteinase k + PMSF 102 + 4~~

~~Heat treatment (2 min at 95°C) 1~~

~~Neuraminidase (0.5 unit/ml):~~

~~Clostridium perfringens 38 ± 3 28 + 41~~

~~Arthrobacter ureafaciens 28 ± 1 o-L-fucosidase (0.5 unit/ml) 90 + 3? N-acetyl-glucosaminidase (0.5 unit/ml) 102 + 8~~

~~Sialic acid (3mM) (92 + 12)~~

~~Gangliosides (lmg/ml) 106 ± 20 (72 ± 14) 53 ± 23~~

Synaptosomes were heat-treated or preincubated in Krebs-phosphate buffer, in the presence or absence of various reagents listed above, using the conditions detailed in Methods. After termination of preincubation by dilution and centrifugation, synaptosomal pellets were resuspended and then incubated with 0.2nM 12*I-BoNT (A) for 40 min at 37°C; binding was quantified by the centrifugation assay given earlier (see Section 4.2.5). Specific binding was calculated by subtraction of non-specific binding (determined for each sample in the presence of ljiM native type A BoNT from total binding and expressed relative to that of the control (± SD, n=3). Figures in brackets are from experiments in which the competing compound was present during both the preincubation and the subsequent reaction of the membranes with 125I-BoNT (A).

* Following enzymic digestion, synaptosomes were incubated with lOnM 125i-Bo NT. 2 Following enzymic digestion, synaptosomes were incubated with InM 125I-Bo NT. 3 1 oc Gangliosides were preincubated with 0.2nM I-BoMT (40 min at 37°C) prior to incubation with synaptosomes (40 min at 37°C). - 186 -

~~Fig. 5.1 Heat and trypsin sensitivity of neuronal acceptor(s) for~~

~~125I-Bo NT (B).~~

~~Synaptosomal membranes (50-lOOpg protein) were preincubated with~~

~~O.lmg/ml trypsin at 37°C (A) or heated at 95°C (B). After various~~

~~intervals, preincubation was terminated by either cooling on ice (B) or~~

~~dilution with buffer containing an excess of trypsin inhibitor and~~

~~centrifugation (A). Binding to treated membranes was initiated by the~~

~~addition of type B *^I-BoNT; (A) InM final concentration or (B)~~

~~0.5nM (O) and lOnM (•) final concentration. B^ and Bto represents~~

~~specific toxin binding at time t and time zero (untreated membranes)~~

~~respectively.~~

* - 187 -

~~Fig. 5.1~~

♦

~~B 0 o 10 20 min - 188 -~~

~~In view of the possible involvement of a component containing~~

~~sialic acid in the action of BoNT (cf.Simpson, 1981b), the sensitivity~~

~~of the binding components to treatment with pure preparations of~~

~~neuraminidases was tested. Incubation of synaptosomes for 40 min at~~

~~♦ 37dC with neuraminidase significantly decreased (60-70%) the amount of 125 type A I-BoNT specifically bound as measured with 0.2nM and lOnM~~

~~labelled toxin (Table 5.1); a similar decrease in binding was shown~~

~~using InM type B *^I-BoMT (Fig. 5.2). This inhibition of types A and * 125 B I-BoNT binding was dose-dependent (0-0.1 unit/ml) under the~~

~~conditions used (Fig. 5.2); the specificity of this inhibition was~~

~~demonstrated as preincubation of neuraminidase with 2, 3-dehydro-2-~~

* deoxy-NANA (a specific inhibitor of neuraminidase) prior to the addition

~~of synaptosomes prevented any effect of this enzyme on toxin-binding~~

~~(Fig. 5.2). These results suggest some involvement of sialic acid~~

* residues in both the high- and low-affinity binding sites for type A 125 I-BoNT and at least in the high-affinity sites for type B toxin.

~~However, the presence of exogenous sialic acid (to 3mM) had no~~

~~appreciable affect on the binding of 0.2nM (Table 5.1) type A *^ I-~~

~~BoNT to synaptosomal membranes. The presence of a mixture of ganglio-~~

sides in the assay mixture was found to lessen the ability of type A 125 I-BoNT to bind synaptosomes, but this reduction was small U30%) « even at high ganglioside concentrations (lmg/ml) (Table 5.1 )y. fn

~~addition, preincubation of synaptosomes with gangliosides failed to~~

~~alter the binding of type A I-BoNT (Table 5.1). However, a 125 4 significant effect on type B I-BoNT binding was observed when toxin~~

~~(1.3nM) was preincubated with gangliosides (40 min at 37°C) before the~~

~~addition of synaptosomes; under these conditions there was about a 60%~~

~~reduction in binding (Fig. 5.3).~~

~~The involvement of sugar residues in toxin binding was~~

~~investigated using a variety of lectins. Following incubation of the - 189 -~~

~~Fig. 5.2 Effect of neuraminidase on synaptosomal binding of types A and~~

~~B 125I-Bo NT.~~

♦

~~i t~~

~~Neuraminidase (0-0.1 units/ml) from Cl. perfringens was incubated~~

~~(20 min at 37°C) in the presence (open symbols) or absence (closed~~

~~symbols) of 20mM 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (an~~

~~inhibitor of neuraminidase), after which synaptosome suspensions were~~

~~added. Enzyme digestion was terminated by centrifugation and washing of~~

~~the synaptosomal pellet with Krebs-phosphate buffer containing albumin~~

and the above neuraminidase inhibitor. Toxin binding was initiated by * 125 resuspending the pellet in buffer containing 0.5nM type A I-BoMT 125 (open and closed circles) or InM type B I-BoNT (open and closed

~~squares) and terminated after 40 min at 37°C by dilution and~~

~~centrifugation. BL and BLQ represent the amount of specific toxin~~

~~binding observed after and prior to treatment of membranes with~~

~~neuraminidase. Specific binding was calculated as described elsewhere. 125 Fig. 5.3 Interaction of type B I-BoNT with gang!iosides.~~

A mixture of gangliosides at various concentrations (0-lmg/ml, 125 final concentration) were incubated with 1.3nM type B I-BoNT for 40 min at 37°C after which toxin binding was examined by the addition of synaptosome suspensions and a further incubation, 40 min at 37aC.

Binding was terminated by dilution and centrifugation as described previously; pellets were washed once before submitting to * -radiation counting. Specific binding was obtained by subtracting non-specific from total binding, as detailed elsewhere. Non-specific binding was determined in the presence of several different concentrations of 125 ganglioside. BL and BLQ represent the amount of specific I-BoNT binding in the presence and absence of ganglioside, respectively. - 191 -

~~Fig. 5.4 Effect of lectins on the binding of *2^I-BoNT (A and B).~~

~~Lectins were preincubated with synaptosomal membranes for 60 min~~

~~at 22°C [Concanavalin A (A)]or 4°C [Soyabean agglutinin (A), Ricinus~~

~~communis aggluti nin (□ ) and wheat germ aggluti nin (• and O )]. The pre-~~

~~incubation was terminated by dilution with ice-cold buffer and~~

~~centrifugation; after resuspension in Krebs-phosphate buffer containing~~

~~albumin (at 4°C), binding was initiated by the addition of A) type A~~

~~12^I-Bo NT or B) type B 125I-BoNT, both to a final concentration of~~

~~InM. Binding was terminated after 40 min at 4°C and specifically bound~~

~~toxin calculated as previously described. Non-specific binding was~~

~~determined in the absence and presence of each lectin used. The~~

~~specificity of inhibition observed by wheat germ agglutinin in A) and B)~~

~~was demonstrated by incubation of this lectin in the presence of excess~~

~~N-acetyl-glucosamine (o). BL and BLQ represent the amount of specific~~

~~toxin binding in the presence and absence of lectin, respectively.~~

♦ BL/BL 5.4Fig. 192 - 192 latter with synaptosomal suspensions (60 min at 4°C or 22°C) and removal 125 of free lectin by washing, the binding of InM I-BoMT (A and B) was studied at 4°C. The most marked effect was shown by WGA, which inhibited 1 25 70 and 90% of the specific binding of type A and B I-BoMT, respectively (Fig. 5.4). Also, RCAj was found to prevent both toxins from binding, although with lower efficacy. Neither Con A or SBA had 125 any noticeable effect on the binding of type A or B I-BoNT (Fig.

5.4). The inhibition by RCAj may be due to an indirect effect of some nature since incubation of synaptosomes with 8-galactosidase, specific for internal 8-galactose residues, did not affect the subsequent 125 specific binding of type A or B I-BoNT to its high- or low- affinity sites (data not shown). However, it is possible that owing to the large size of the enzyme (Mr=464000), access to its site of action may be prevented. The significant reduction in toxin binding caused by low concentrations of WGA is specific as preincubation of the lectin 125 with excess NAG prevented any later inhibition of I-BoNT binding

(Fig. 5.4) to synaptosomes. Furthermore, this inhibition is thought to be due to an interaction with NANA (sialic acid) rather than NAG, as preincubation of synaptosomes with N-acetyl glucosaminidase had no effect on the binding of type A *^I-BoNT (Table 5.1). Another enzyme, a-L-fucosidase, tested for its ability to interact with acceptors for type A I-BoNT was without effect (Table 5.1).

Collectively, these findings suggest that sialic acid containing moieties (which may be specific gangliosides) in association with proteinaceous components, appear responsible for the majority of 125 specific I- BoNT binding observed. However, there appears to be a 125 population of sites for type B I-BoNT that are insensitive to protease treatment; in addition, there also exist heat-resistant acceptors. However, whether or not these populations of resistant sites are one and the same requires further examination; preliminary Fig. 5.5 Target size analysis of acceptors for 125I-Bo NT (A and B).

A linear accelerator was used to treat lyophilised synaptosomal membranes with high-energy radiation. Synaptosomal membranes (in the presence of B-galactosidase) and various enzymes (as molecular weight markers) were lyophilised in pyrex glass tubes, sealed under vacuum and stored at -80°C prior to irradiation. Samples, placed in an aluminium holder and cooled in a dry ice/acetone bath were irradiated for various times at a dosage of 0.4 Mrad/min. Membranes were resuspended in 5mM sodium phosphate buffer, pH 7.5, containing Img/ml bovine serum albumin and aliquots taken for analysis of enzyme activities and toxin binding.

~~The latter was assayed by incubation of membranes with either 2nM type A~~

*^I-BoNT (•) or 8.3nM type B *^I-BoNT (■) for 40 min at 22°C; specific binding was determined as described earlier. Enzyme standards used were B-galactosidase (Mr 464000, □ ); pyruvate kinase

(Mr 224000*4); phosphocreatine kinase (Mr 170000,A) and alcohol dehydrogenase (Mr 84000,0). A) A and AQ represent the toxin binding or enzyme activity of a sample, after and before irradiation, respectively. B) Calibration plot for inactivation of enzyme markers of known molecular weight. and represent the molecular weights

~~(Mr) of each standard enzyme and B-galactosidase, respectively; IQ is the ratio of the inactivation rate of enzyme standards in relation to that of B-galactosidase. - 195 -~~

~~Fig. 5.5~~

~~Mrad - 196 -~~

~~experiments (not shown in this report) indicate that the answer to this~~

~~problem is not straightforward. To date, the differential sensitivities~~

~~to heat and protease treatment are the only anomalies observed in the 125 nature of the synaptosomal acceptors for types A and B I-BoNT.~~

~~♦ 125 5.3.2 Target size analysis of acceptors for I-BoNT (A and B). 125 Attempts to covalently cross-link I-labelled neurotoxins to their~~

~~synaptosomal acceptors using bifunctional reagents (e.g. dimethyl -~~

~~suberimidate, dimethyladipimate; Smith and Loh, 1978) have so far been~~

~~unsuccessful. Therefore, to gain some insight into the molecular size~~

~~of such acceptors, irradiation inactivation studies on freeze-dried~~

* membranes were performed. Membrane samples and enzyme standards were

~~subjected to a high-energy electron beam (7.IMeV) produced by a linear~~

~~accelerator for dosages ranging from 3-15 Mrad. After resuspension,~~

~~samples were tested for their enzyme activities and ability to * 125 specifically bind types A and B I-BoNT. This process gave a~~

~~decrease in the activities of enzyme standards relative to their~~

~~molecular weights (Fig. 5.5); however, no effect on toxin binding was~~

~~observed using low or high concentrations of types A or B ^ I - B o N T ,~~

~~respectively (Fig. 5.5). Thus the target size of these high- and low- J affinity acceptors located on synaptosomal membranes is very small; from~~

* the molecular weight calibration plot (Fig. 5.5, B) if 15 Mrad of

~~irradiation resulted in a 10% decrease in specific binding this would~~

~~indicate a Mf of <20000 for the functional acceptor(s). These data,~~

~~therefore, suggest the involvement of glycolipids or glycopeptides in ♦ BoNT binding.~~

~~5.3.3 Selectivity of synaptosomal acceptors for types A and B 125I-~~

~~BoNT. Previously a test of the effects of individual subunits from type~~

~~A BoNT on the high-affinity binding of BoNT (A) to its acceptors) has - 197 -~~

~~Fig. 5.6 Effect of type B BoNT and subunits from type A BoNT on~~

~~BoNT (A) binding.~~

125 Type A I-BoNT (InM, final concentration) was added to increasing concentrations of test compound. 3inding was initiated by the addition of synaptosome suspension (0.5mg/ml, final concentration) and terminated after 40 min incubation at 37°C by dilution and centrifugation as described elsewhere. BL and BLq represent the 125 amount of type A I-BoMT binding in the presence and absence of competing ligand; unlabelled type A BoNT (o), 55000 - M p (0) or 91000

~~- Mr (■) subunit of type A BoMT, type B BoNT (♦). - 198 -~~

~~Table 5.2 Selectivity of the high-affinity acceptor(s) for 125I-Bo NT~~

~~(A and B> on synaptosomal membranes.~~

~~Relati ve specific Toxin Treatment binding [%)~~

125I-BoNT (A) None (total binding) 100 ± 9 Type B BoNT (lyM) 97 ± 9 Type B neurotoxin - * haemagglutinin complex (lmg/ml) 99+4 Tetanus toxin (lpM) 62 ± 3 Crotoxin (luM) 0 11 ± 2 Crotoxin (luM) (-Ca‘+ ; ImM EDTA) 67 ± 10 Taipoxin (luM) 9 4 ± 1 Ta1pox1n (luM) (-Ca^; lmM EDTA) 91 ± 12 Bee venom phospholipase A« (luM) 1 ± 0.1 Bee venom phospholipase A^ (lum) (-Ca2+; ImH EDTA) 100 ± 0.3

~~125I-Bo NT (B) None (total binding) 100 ± 7 Tetanus toxin (lpM) 49+3 Taipoxin (luM) 3 ± 0.2~~

~~None (total binding, at 4°C) 100 ± 8 Taipoxin (luM) (at 4®C) 93 ± 4~~

~~♦ Synaptosomes were preincubated in Krebs-phosphate buffer in the~~

~~presence or absence of various reagents listed above for 40 min at 37°C~~

~~(unless otherwise stated). After termination of preincubation by~~

~~dilution and centrifugation, synaptosomal pellets were resuspended and~~

~~then incubated with either 0.2nM *^I-BoNT (A) or 0.5nM *^I-BoNT (B)~~

~~for 40 min at 37°C (unless otherwise stated); binding was quantified as~~

~~described earlier. Specific binding was calculated by subtraction of~~

~~non-specific binding (determined for each sample in the presence of luM~~

~~native BoNT (A or B)) from total binding and expressed relative to that~~

~~of the control (±SD, n=3). not been feasible. This is now possible using I-BoNT (A) and the subunits of type A BoNT separated and renatured as described earlier~~

(see Chapter 2). Individual toxin subunits (after dialysis against 25mM sodium phosphate buffer, pH 7.5, containing 0.1M NaCI) were tested for 1 25 their ability to compete with the binding of type A I-BoNT (Fig.

~~5.6). The larger subunit (Mr 91000) was effective in preventing the binding of A I-BoNT (A) and was only slightly less potent than native~~

BoNT (IC5q of 5 x 10‘8M and 3 x 10“8M respectively); the ICgQ is that concentration of competing ligand which displaces 50% of the specific binding of labelled toxin. This discrepancy in the IC^q values obtained between experiments is probably due to the different 125 preparation and concentrations of I-BoNT and synaptosomes used.

~~The smaller subunit (Mr 55000) retained about 1% of the specific neurotoxicity of the native protein (compared with 0.1% for the larger subunit; see Chapter 2) but it failed to inhibit the binding of~~

125 I-BoNT (A) at concentrations up to lpM. However, it must be noted 125 that the high-affimty sites for I-BoNT are not distinguished in the type of competition assay described above; hence, the inhibition observed by the H-subunit (above) and various other ligands (described later in this section) may represent an effect solely on the low-affinity acceptors.

To investigate the possible presence of homology between the neurotoxin binding component(s) for the different botulinal types, the ability of type B BoNT (Table 5.2; Fig. 5.6) or its complex with haemagglutinin (Table 5.2) to compete for the binding component(s) of 125 type A I-BoNT was studied. Neither high concentrations of type B toxin complex (lmg/ml) nor neurotoxin (up to luM) appeared to have any 125 effect on the binding of I-BoNT (A) to synaptosome suspensions.

~~The binding of type B BoNT to the small percentage of high-affinity sites present was excluded as a lack of inhibition was seen when the - 200 -~~

~~Fig. 5.7 Effect of nicked BoNT (B), BoNT (A), B-bungarotoxin and~~

~~dendrotoxin on binding of type B ^^I-BoNT to synaptosomes.~~

~~Type B I-BoNT (InM, final concentration) was added to A increasing concentrations of test compound and incubated with~~

~~synaptosome suspensions as described earlier (Fig. 5.6, legend):~~

~~unnicked type B BoNT (O); nicked (trypsin, 5ug/mg, 30 min at 22°C )~~

~~type B BoNT (■); type A BoNT (#); dendrotoxin (□); B-bungarotoxin~~

~~(A). BL and BLQ are as defined in Fig. 5.6. - 201 -~~

~~Fig. 5.8 Effect of tetanus toxin, 8-bungarotoxin, dendrotoxin and 125 botulism antagonists on the binding of I-BoNT (A).~~

~~1 pc Type A I-BoNT (2nM, final concentration) was added to~~

~~increasing concentrations of test compound: native type A BoNT (O);~~

~~tetanus toxin (A); 8-bungarotoxin (v); dendrotoxin (■); 4-amino-~~

~~pyridine (□). Binding was initiated by the addition of synaptosome~~

~~suspension and terminated after 40 min at 37°C as previously described.~~

~~BL and BLQ represent the amount of I-BoNT (A) binding in the~~

~~presence and absence of the aforementioned competing ligands. - 202 -~~

~~125 experiment was performed using 0.2nM type A I-BoNT (Table 5.2). In~~

~~contrast, type A BoNT, at high concentrations ( > 5 x lCf^M) showed a 125 slight inhibition of type B I-BoNT binding to synaptosomes (Fig.~~

~~5.7); however, the low affinity of such an interaction casts doubt on~~

* its physiological significance.

~~As mentioned earlier (Chapter 2), type B BoNT may exist as a~~

~~single (partially active) or dichain (fully-active) molecule; the~~

latter being obtained in vitro by mild trypsinisation of the single- w o chain form. The dichain molecules are more potent (2 x 10 mouse o LDgg/mg) than the unnicked toxin (^10 mouse LDgQ/mg); at present

~~it is unknown whether this increase in toxicity is due to an increased~~

~~affinity of the neurotoxin for its acceptor(s) or an effect at a~~

~~subsequent stage in the toxin’s action. This was investigated by 125 studying the binding of InM type B I-BoNT in the presence of~~

~~increasing concentrations of native (mainly single chain molecules) or ♦ trypsinised (fully active,dichain molecules) type B BoNT (Fig. 5.7).~~

~~Under the conditions used there was no significant change in the binding~~

~~affinity of these two forms of BoNT for its acceptors)suggesting an * effect of nicking in a later phase of the toxin's action.~~

~~The possibility that BoNTs share their binding components with~~

~~other presynaptic toxins was tested by measuring their ability to~~

~~♦ compete with I-BoNT (A and B) for these sites. Purified tetanus~~

~~toxin, another clostridial neurotoxin from Cl. tetani, was found to 125 antagonise the specific binding (Fig. 5.8) of type A I-BoNT, albeit~~

~~% with low efficacy (IC,jq = 750nM) and hence of questionable physio logical significance. Likewise, high concentrations of tetanus toxin~~

~~were required to displace I-BoNT (A or 3) binding when low~~

~~concentrations of the latter (0.2nM or 0.5nM, respectively) were used~~

~~(Table 5.2). B-bungarotoxin and dendrotoxin had no significant effect~~

~~on type A or B I-BoMT binding (Figs. 5.7 and 5.8); also a- latro- - 203 -~~

~~Fig. 5.9 Ability of a-latrotoxin, crotoxin, taipoxin and bee venom 125 phospholipase Ap to interfere with I-BoNT (A) binding.~~

~~125 I-BoNT (InM, final concentration) was added to increasing~~

* concentrations of unlabelled type A BoNT (O) or to one of a number of

~~test compounds. Binding was initiated by the addition of synaptosome~~

~~suspension and terminated after 40 min at 37°C in the presence of~~

~~^ Cac , except where specified, by dilution and centrifugation. BL and 125 BLq represent the amount of type A I-3oNT binding in the presence~~

~~and absence of competing substance. (A) a-latrotoxin (□); taipoxin~~

~~(▼); taipoxin, minus Cac (v); native type A BoNT, 4°C (•); taipoxin, * 4°C (■). (B) Bee venom phospholipase A2 (▼); crotoxin (V); bee venom~~

~~phospholipase, minus Ca (■), the inhibition seen at high~~

~~concentrations could result from reduced enzymic activity or steric~~

* hindrance.

* fo O

~~Fig. 5.9 0 BL/BL 4 4~~

~~-log[Ligand] (M) ♦ * * BL/BL0~~

-loglLigand] (M) toxin (up to 20nM) was unable to inhibit the synaptosomal binding of type Ipc A " I-BoNT (Fig. 5.9). Taipoxin, crotoxin and bee venom phospholipase 195 A2 , inhibited the binding of type A “ I-BoNT to its low- (Fig. 5.9) and high- (Table 5.2) affinity sites; a similar inhibition of type B 125 I-BoNT binding to its high affinity acceptor(s) by taipoxin was also observed (Table 5.2). The large diminution in their inhibitory effects on the binding by lowering the incubation temperature to 4°C (Table 5.2,

~~Fig. 5.9A) or removal of Ca2+ (Table 5.2, Fig 5.9B) suggests their phospholipase activities were responsible for the apparent antagonism.~~

~~Certain compounds (e.g. chloroquine, theophylline and 4-amino- pyridine) have been reported to delay onset or temporarily reverse the~~

~~physiological actions of BoNT (Howard et al_., 1976; Dreyer and Schmitt,~~

~~1981; Simpson, 1982; Ball et , 1982). Neither 4-ami nopyridine (Fig.~~

5.8) nor any other such antagonist (chloroquine, theophylline, methyl amine, ammonium chloride) affected the binding of type A 125 I-BoNT to synaptosome suspensions in routine competition assays which tends to suggest that their effects are exerted at a location(s)

~~distinct from the original binding site(s).~~

~~125 5.3.4 Preliminary studies on the preparation of I-labelled subunits~~

~~from BoNT (A). In this short study, two aspects were looked at; firstly,~~

~~the electrophoretic characteristics of the labelled subunits of type A 125 I-BoNT were investigated using two-dimensional PAGE and secondly,~~

~~attempts were made to prepare labelled H-subunit from type A BoNT by~~

~~direct radioiodination. To determine the pi of the individual subunits~~

~~from I-BoNT (A), the latter was subjected to two-dimensional PAGE~~

~~under denaturing conditions in the presence of reducing agent. An auto~~

~~radiogram of the dried SDS-gel showed a distinct separation of heavy and 125 light subunits from intact type A I-BoNT (Fig. 5.10). The position 125 Fig. 5.10 Two dimensional IEF-SDS PAGE I-BoNT (A).~~

~~125 I-BoNT was subjected to narrow range (pH 5-8) isoelectric~~

~~focussing in the first dimension, performed under denaturing conditions~~

(8M urea) in the presence of 5% 2-mercaptoethanol. After focussing, the gel was sliced longitudinally; one half was used for determination of the pH gradient whilst the other half was equilibrated (30 min) in SDS- gel electrophoresis sample buffer containing 2-mercaptoethanol before application to a 10% SDS-polyacrylamide gel. The focussed gel was sealed in place with Agarose (1% w/v in the above sample buffer); radiolabelled molecular weight standards were run in a track near the side of the gel. Following electrophoresis, the gel was fixed (25% isopropanol/10% acetic acid), washed thoroughly with deionised water, dried and submitted to autoradiography. The autoradiogram of the dried gel is shown. Note the separation of heavy and light subunits from intact toxin. - 207 -

~~125 Fig. 5.11 Specific binding to synaptosomes of I-labelled subunit~~

~~(M_ 91000) from BoNT (A). — r ------~~

~~125 I-labelled subunit (M„ 91000), InM final concentration, was r added to increasing concentrations of the unlabelled subunit. Binding~~

~~was initiated by the addition of synaptosome suspension and terminated~~

~~after 40 min at 37°C by dilution and centrifugation. BL and BLq~~

~~represent the amount of labelled-subunit binding in the presence and~~

~~absence of unlabelled subunit. Note the high percentage of non~~

~~specific binding present as compared to that found with intact type A~~

~~^ I - B o N T (see Fig. 5.6, curve for native type A BoNT). - 208 -~~

~~125 of intact I-BoNT (A) was evident from the small amount of non-~~

~~reduced toxin remaining at a higher molecular weight than the large~~

~~subunit. The large and small subunits had pi's of approximately 6.6 and~~

~~6.4 respectively, whilst that of intact toxin was about 7.0. The~~

~~% findings suggest that these three labelled species (intact toxin and~~

~~both of its subunits) may be separated according to their charge, if a~~

~~high resolution technique can be developed which allows their~~

~~solubilities to be maintained (cf. Chapter 2). * . 125 The preparation of a I-labelled derivative of the large~~

~~subunit from type A BoNT that retains its ability to interact with~~

~~subunit was obtained by QAE-Sephadex ion-exchange chromatography (as~~

~~described in Chapter 2); after renaturation by dialysis against 125 sodium-phosphate buffer it was labelled with I-iodine, as described~~

* for type A BoNT (see Chapter 4), to high specific activity (920 Ci/mmol). However, when tested in a competition assay with unlabelled

~~subunit (Fig. 5.11), the amount of non-specific binding was very high~~

~~(>60%) as compared to that found with type A I-BoNT (see Fig 5.6, * curve for native type A BoNl). In addition, this preparation was~~

~~relatively unstable upon storage at 4'C; specific binding was only~~

~~detectable for 2-3 days after radiolabelling as compared with 2-4 weeks ♦ 125 for I-BoNT preparations.~~

~~5.4 DISCUSSION~~

~~125 Successful preparation of I-BoMT (A and B) to high specific~~

~~radioactivity has produced an extremely sensitive probe which has been~~

~~used for the first time to disclose the nature and selectivity of its~~

specific high and low-affinity acceptors on synaptosomal membranes. 125 These membrane-bound acceptors for I-BoMT (A) are thought to be of a proteinaceous nature since their activity is completely removed after proteolysis with proteinase k or trypsin, or by heating. However, 125 acceptors for type B I-BoNT showed only partial sensitivity to protease and heat treatments suggesting the involvement of a non-proteinaceous component(s) in some of the binding observed. At least in the case of the heat-resistant sites, these appear to be of high affinity, as the binding of low concentrations (0.5nM) of type B 125 I-BoMT to heat-treated synaptosomes remained unaffected.

Neuraminidase treatment of synaptosomes reduced significantly the 125 extent of I-BoNT (A and B) binding to the high-affinity components; this inhibition was also shown for the low affinity binding of type A 125 I-BoNT to its acceptors. The failure of protease inhibitors, present during the incubation, to prevent this inactivation shows that it is not due to an effect of proteases that might possibly be contaminating the neuraminidase preparations used; however, the pH of the buffer used may not have been optimal for all the inhibitors present. A specific action of neuraminidase was shown conclusively when a specific neuraminidase inhibitor was found to prevent the inhibitory effect of this enzyme on synaptosomal binding of types A and B 125 I-BoNT. As incubation of the synaptosomes with sialic acid showed 125 no significant effect on type A I-BoNT binding, it is unclear whether these residues interact with the toxins directly. However, binding properties of simple sugars in solution may differ enormously from their properties in a complexed immobilised (e.g. membrane-bound) state (Lotan and Nicolson, 1979). On the other hand, they may be critical in maintaining the conformation of component(s) that may be more directly responsible for toxin binding.

The involvement of sugar residues in the high-affinity binding of 125 types A and 3 I-BoNT was investigated using a variety of lectins. Of those tested, only WGA (specific for sialic acid and NAG residues) and RCAj (specific for 3-galactose) had any significant effect.

Neither N-acetylglucosaminidase or 3-galactosidase affected the binding 1 9R of type A I-BoNT to synaptosomes, indicating the effect of WGA to be on sialic acid residues and that of RCAj to be an indirect inhibit ion of toxin binding. It is, of course, possible that the afore mentioned enzymes could not reach their substrates because of steric hindrance or that the specific substrates in question (NAG and lactose) could not be cleaved due to protection afforded by adjacent saccharides.

Conditions described previously (Cuatrecasas, 1973b) for incorporation of gangliosides into synaptosomal membranes showed no 125 increase in binding capacity for types A I-BoNT, thereby indicating that these gangliosides are not directly involved in the specific binding observed. The partial inhibition of I-BoNT (A and B) binding produced by preincubation of high concentrations of gangliosides 125 with I-BoNT,before the addition of synaptosomes, probably results from interaction of ganglioside with the toxin. Several explanations for such inhibition may be put forward; for instance, the toxin may interact non-specifically with the gangliosides or a specific ganglioside(s) present in the mixture in very small amounts may exert a high-affinity interaction with the toxin. Otherwise, it is possible that gangliosides at high concentrations exhibit a detergent-like effect on the toxin thus preventing it from binding as discussed by van

~~Heyningen and Mellanby (1973).~~

The membrane lipid environment is also shown to be of some 125 importance in the binding of types A and B I-BoMT, since these interactions were inhibited by phospholipase A2 activity as seen with bee venom phospholipase, crotoxin and taipoxin. High-affinity sites for types A and B BoNT were affected in addition to the low-affinity sites (as shown in competition experiments) for type A neurotoxin.

Interestingly, another phospholipase from Maja n. melanoleuca failed to 125 have an effect on the binding of type A I-BoNT to synaptosomes, which may be a reflection of the different substrate specificities of the phospholipases. Similarly, the binding of 3-bungarotoxin and dendrotoxin to their synaptosomal acceptors are inhibited by crotoxin, taipoxin and bee venom phospholipase in the presence, but not in the absence of Ca^+ (Othman et al_., 1982; Dolly et al_., 1984b); this also suggests a non-specific effect of membrane perturbation on toxin binding. Interestingly, as shown for 3oNT binding above, phospholipase from Naja n. melanoleuca (in the presence of Ca ) was also unable to affect the specific synaptosomal binding of 3-bungarotoxin or dendrotoxin.

The functional size of the specific synaptosomal acceptors for 125 I-BoNT (A and B) was investigated using the technique of irradiation inactivation. However, dosages of radiation up to 15 Mrad failed to give any inactivation of the membraneous acceptors, as shown by the binding of 2.0nM type A ^ I - B o N T or 8.3nM type B * ^ I - BoMT. 125 Similar data was found using low concentrations of type B I- BoNT

(InM), suggesting that there is no significant difference in the target size of high- or low-affinity acceptors for this toxin. The activities of internal enzyme standards, irradiated at the same time, decreased at rates relative to their oligomeric sizes. Extreme caution must be exercised in calculation of such calibration plots, as the enzymes used may not always inactivate according to their oligomeric sizes, depending upon the conditions used for sample preparation. For example, if 3- galactosidase is prepared for irradiation in low ionic strength buffer or water, this enzyme inactivates at rates corresponding to its single subunit form and/or a mixture of higher molecular weight forms (R.S.

~~Williams and A.R. Black, unpublished observations); thus an average value taken for its inactivation rate (together with its oligomeric~~

~~size) would not lie on the straight line of the calibration plot.~~

However, these data presented suggest that the functional size of the 125 binding sites for types A and B I-BoNT are very small, as might be expected of a ganglioside or glycopeptide acceptor. This may explain 125 why attempts to covalently cross-link I-BoNT (A and B) to their acceptors and visualise differences in molecular weights of the free toxin and cross-linked toxin-receptor complex, by SDS-PAGE, have so far been unsuccessful.

~~The neurotoxins produced by different toxigenic strains of Cl.~~

~~botulinum exhibit differential inter-species and intra-species toxicity,~~

despite the fact that they are (with some exceptions) dichain molecules of similar molecular weights (Mr 140000-160000) linked by disulphide bridges (Sugiyama, 1980; Simpson, 1981a). It is unknown whether these distinct characteristics are due to differences in their binding

~~properties or if they result from variations in a subsequent step in~~

their action. Type B BoMT or its complex with haemagglutinin was unable to compete for the high- or low-affinity specific binding sites of type 125 A I-BoNT suggesting that, at least in brain synaptosome preparations, its binding site(s) differ from those for type A BoNT.

~~However, BoNT (A), at high concentrations was found to partially prevent 125 type B I-BoNT from binding to synaptosomes indicating that type A~~

BoNT may share some of the sites for type B BoNT, albeit with low affinity. It is interesting to note that recent results from our 1 25 laboratory show a similar effect of type B BoNT on type A I-BoMT binding and of type A BoNT on I-BoNT (B) binding at the neuro muscular junction (see General Discussion). In addition, some evidence has been reported which supports the data obtained on synaptosomal bind- 125 ing of I- BoNT (Kozaki, 1979), although the binding of type B BoNT to high affinity sites for type A BoNT was not examined. In addition, this worker reported that types A and E BoNT share at least some acceptors with similar (apparently low) affinity, suggesting that not all the different types of BoNT have mutually independent binding sites.

It is well known that some types of BoNT are produced as partially active, single chain molecules which may be converted to the dichain, fully active form by trypsinisation. Using native type B BoNT (which is mainly the single chain form) and nicked type B BoNl, their abilities to compete for the binding sites of I-BoNT (B) were studied; no significant differences were observed in the binding affinities of the two forms for their acceptors indicating that the binding step is not the phase in the toxin's action where this difference in structure is of importance. A similar result was reported from equilibrium binding 125 experiments with a I-labelled derivative of type E BoNT (Kozaki and

~~Sakaguchi, 1982); owing to the methodologies employed in this study and the high content of very low- affinity sites for this toxin preparation~~

(Kd % lOOnM, Bmax % 20 pmol/mg protein),this result remains at least questionable. Moreover, these studies are concerned with low-affinity acceptors; to date no study has been made on the effect of nicking on the toxin's ability to bind to high-affinity site(s).

~~In an attempt to assign functions to the individual subunits of~~

~~BoNT, the polypeptides of type A BoNT were separated and their effect on the binding of I-BoNT (A) to synaptosomal membranes was examined.~~

The observed ability of only the larger of the two subunits to inhibit 125 I-BoNT (A) binding, suggests that it is directly responsible for the binding. The slightly lower affinity of this subunit for the binding component(s) may be due to conformational changes that occurred on reduction of the intermolecular disulphide bridge(s) and subsequent separation of the polypeptides. Dolly et al_. (1984a) recently reported 125 that the binding of this I-BoNT (A) preparation to its specific acceptors at the neuromuscular junction is also mediated by the larger subunit. In the case of type B BoNT, its heavier subunit was found to bind to brain synaptosomes with the same affinity as that of the native toxin (Kozaki, 1979); in addition, the lighter polypeptide exhibited appreciable, but lower, affinity for these sites, probably due to it being contaminated. It has been proposed that the action of botulinum toxin is brought about by a multi-step process involving initial extra cellular binding, translocation and a paralytic step (Simpson, 1980) as previously shown to exist for other microbial and plant toxins (Collier,

1975; Olsnes and Pi hi, 1977; van Heyningen, 1977). The ability of the larger subunit to bind, despite its lack of toxicity, is direct evidence that additional step(s) must be involved in the toxin’s action. The lack of inhibition of binding by the smaller subunit suggests it might contribute to one or more of the subsequent phases. Tetanus toxin was found to inhibit only partially the binding of type A I-BoNT to its high-affinity and low-affinity sites on brain synaptosomes, even at relatively high concentrations (luM). A similar inhibition of type B 175 ‘ I-BoNT binding to its high affinity site(s) was also found. Hence, it seems unlikely that this is the primary site for tetanus toxin binding as its Kj would be inconsistent with its high potency, at least in the central nervous system. Also, a certain lack of specificity appears to exist in these interactions since tetanus toxin inhibits both BoNT (A and B) binding even though the sites for the latter toxins are mostly independent. The absence of high-affinity and the presence of low-affinity acceptors for tetanus toxin at the NMJ

(i.e. the acceptors for BoNT) may help to explain the much decreased effectiveness U 500-1000 times less potent than BoNT) of tetanus toxin at peripheral synapses (Habermann, 1981). It is of course possible that tetanus toxin affects I-BoMT (A and B) binding indirectly through conformational changes or steric hindrance, thus inhibiting both type A and B BoNT binding without necessarily interacting with either of the toxin's acceptor(s). However, it is interesting to recall that the

~~binding activity of tetanus toxin resides in the larger of its two sub~~

~~units (van Heyningen, 1976). Other presynaptically-active neurotoxins~~

~~tested in their ability to interact with specific sites on synaptosomal membranes were 3-bungarotoxin and dendrotoxin. Both of these toxins~~

~~have been shown independently to bind saturably to rat brain synaptic membranes (Othman et al_., 1982; Rehm and Betz, 1982; Dolly et al.,~~

1984b). Neither toxin affected the binding of type A or B *^I-BoNT to synaptosomes suggesting these toxins have distinct sites. In support of this conclusion, type A BoNT did not inhibit the binding of a

~~radio-labelled B-bungarotoxin derivative to synaptosomal membranes~~

~~(Othman et al., 1982).~~

IOC The larger subunit of type A BoNT was labelled with I-iodine in an attempt to utilise this protein as a non-toxic probe to investigate properties of components specifically located on the presynaptic membrane and their role in toxin internalisation. This 125 I-subumt was of high specific radioactivity, but when tested in its ability to bind to synaptosomes showed a high degree of non-specific binding; in addition, this preparation was relatively unstable upon storage, usable for a few days rather than weeks (as with intact 125 I-BoNT. This tends to suggest that unless further refinements are made to the iodination procedure used, this method of achieving a highly radioactive preparation of the larger subunit from type A BoNT is not viable. However, this is the first direct evidence that the large subunit of BoNT binds specifically to neuronal membranes. An alternative approach to the preparation of I-labelled subunits from BoNT (A) would be to separate these from the intact toxin after radioiodination.

The two ^^I-subunits from ^^I-BoNT (A) were found to have different pis as illustrated by two-dimensional PAGE. Therefore, once the solubility problems of the lighter subunit have been overcome (as - 216 - ' discussed in Chapter 2), it may be possible to separate these subunits on a preparative scale by a chromatographic procedure utilising their differences in charge.

In summary, these findings indicate that the specific binding of types A and B BoNT to synaptosomes (which is mediated by the larger sub unit is due to a sialic acid-containing moiety (possibly a specific ganglioside) in direct or indirect association with a proteinaceous component. If the association is direct, then a small glycopeptide rather than a ganglioside is probably involved, owing to the small target size of the acceptors.

The acceptors for types A and B BoMT are very similar in nature and their selectivity for ligands. However, the partial sensitivity of acceptors for type B BoNT to treatment with trypsin or heat (unlike the absolute effect on type A BoNT acceptors), must be stressed; one must also be reminded that the majority of sites for types A and B neurotoxins are independent of each other.

~~In the following chapter the functional significance of such specific acceptors for BoNT (A and B), as described above and in Chapter~~

~~4, is discussed in relation to these toxins' actions on the mechanism(s) of transmitter release. - 217 -~~

~~CHAPTER 6~~

~~GENERAL DISCUSSION~~

~~♦ - 218 -~~

~~6.1 BOTULINUM NEUROTOXINS - AN HOMOLOGOUS GROUP.~~

~~The properties of botulinum toxins have many aspects which are of~~

~~widespread scientific interest. Although botulism does not occur in~~

* epidemic proportions nowadays, minor outbreaks are still relatively

~~frequent (Lewis, 1981); the clinical aspects have been highlighted in~~

~~recent years with the discovery of a relationship between botulism and~~

~~some sudden infant cot deaths (Arnon, 1981). In relation to human * botulism, the rapid and sensitive screening for the different botulinum~~

~~toxins in clinical samples and foodstuffs is diagnostically and~~

~~commercially of great importance. BoNT shows great specificity in its~~

~~inhibition of ACh release in the peripheral nervous system, where it is~~

~~thought to interfere with an integral part of the release mechanism(s)~~

~~(see General Introduction). Such specificity and unique potency affords~~

* to this group of toxins a considerable potential for elucidation of molecular mechanism(s) involved in transmitter release and also for its

~~possible use as a specific marker for cholinergic synapses; however, in~~

~~some instances, BoNT has been shown to block non-cholinergic synapses *- (Mackenzie et al_., 1982), discussed later.~~

~~To date, eight forms (A through G) of BoNT have been described;~~

~~different strains of Cl. botulinum release these toxins into culture m media as complexes with non-toxic proteins (some of which may exhibit~~

~~haemagglutinating activity). These complexes are thought to protect the~~

~~neurotoxin moieties from denaturation in the digestive tract following~~

~~4 oral ingestion (Ohishi et al_., 1977; Sakaguchi et al_., 1981). A wide~~

~~variety of chromatographic techniques have been employed in the~~

~~purification of neurotoxin from such complexes, many using a crystalline~~

~~form of type A toxin as a starting material. The affinity chromato~~

~~graphy procedure of Moberg and Sugiyama (1978) was used by Tse et al.~~

(1982) to develop a three step chromatographic purification of type A BoNT from acid precipitates of crude cultures. In Chapter 2, this method was readily applied to achieve a rapid and efficient purification of type B neurotoxin for the first time; the neurotoxin was homogeneous on SDS-PAGE and exhibited a high specific neurotoxicity (1.1 x 10** mouse LDgQ/mg protein). The fact that six out of the eight types of toxin complex are known to contain haemagglutinin proteins, together with the demonstration that types A and B neurotoxin may be purified to homogeneity using the same overall conditions (Chapter 2), suggests that this method may be employed for the purification of all the remaining types of neurotoxin-haemagglutinin complex. However, some of these toxins may have different sugar specificities; type C toxin complex is reported to interact with human erythrocytes (Boroff and DasGupta, 1971) but not chicken erythrocytes (Lamanna and Lowenthal, 1951).

Although the published procedures reported for the separation of neurotoxin subunits are only applicable to a single neurotoxin type, minor modification of the method described in Chapter 3 for the separation of polypeptides from types A and B neurotoxins using HPLC may prove this to be suitable for all types of BoNT. In addition, it is likely that HPLC will prove effective in the preparation of subunits

~~(Mf 'ulOOOOO and 50000) from tetanus toxin; to date, these have been prepared using conventional permeation chromatography (Craven and~~

~~Dawson, 1973; Matsuda and Yoneda, 1975), with concomittant lower recoveries of protein after many hours of chromatography (elution of both subunits requires %20 hr).~~

~~The subunit structure of all BoNT types is very similar, a large~~

~~(Mr -ulOOOOO) and small (Mr ^50000) subunit which, with the exception of type C2 toxin, are linked by a disulphide bond (Sugiyama,~~

1980). Although type E BoNT exists in culture only as unnicked molecules, it may be cleaved by trypsin into the dichain form (DasGupta and Rasmussen, 1981). The observation that in 1-4 day old cultures of Cl. botulinum, neurotoxin is present either solely as the single chain form or as a mixture of single and dichain molecules (excepting type C toxins) suggests that they are probably synthesised as a single polypeptide. type B neurotoxin was purified as a mixture of nicked and unnicked molecules (Chapter 2); this corresponds with an earlier report

(DasGupta and Sugiyama, 1976) that a preparation of type B BoNT from late cultures (4-7 days) contained both forms of toxin. However, presently it is unknown why only a proportion of the molecules are nicked. One possibility is that the enzyme(s) responsible for nicking is synthesised only by older cultures thereby allowing most neurotoxin to be secreted in its single-chain form. However, the latter form of this toxin may be converted to the dichain molecule by trypsinisation

(DasGupta and Sugiyama, 1976) with a concommitant increase in neurotoxicity. The increased potency of this activated toxin is not thought to be due to a greater affinity for its acceptor as it showed no significant increase in its affinity for synaptosomal binding sites over that of mainly unnicked, partially active BoNT (B). This is not an un expected finding since the binding step is not believed to be rate- limiting in the action of BoNT.

Despite the strong similarities in structure, the different types of BoNT remain immunologically distinguishable, although there is an indication of a small degree of cross-reactivity between types E and F

~~BoNT (Eklund et al_., 1967; Yang and Sugiyama, 1975). More recently, using monoclonal antibodies against type toxin, Oguma et al.,~~

~~(1982) have shown some cross-reactivity between types and D neuro toxins. Hence the reports that the individual subunits of types A, B,~~

C^, and C2 BoNT (see Chapter 2; Kozaki and Sakaguchi, 1975; Syuto and Kubo, 1981; Ohishi, 1983) have distinct antigenicities, may be a reflection of the limited types of antibodies presently available. Cl. botulinum types C and 0 contain phages that are known to be responsible for the production of neurotoxin (Eklund et a]_., 1972; Inoue and Iida, 1971). These strains, cured of their tox+ phage, no longer produce or D toxins as a major or minor form (Eklund and Poysky,

~~1974); they may be made fully toxigenic again by reinfection with a lysed extract of either of the parent strains (Inoue and Iida, 1971).~~

These reports suggest that a single phage may be responsible for the production of more than one toxin. However, type toxin, produced by types C and D cultures of Cl. botulinum, is still present when these cultures are cured of their tox+ phage, when they no longer produce

C^ and D toxins; this suggests that the synthesis of C2 toxin is not phage-mediated (Eklund and Poysky, 1972). Despite this anomaly of type C2 toxin, all species of Cl. botulinum have been reported to carry temperate phages (Dolman and Chang, 1972; Inoue and Iida, 1968;

~~Scott and Duncan, 1978); hence, it is plausible that all of the other neurotoxins may have arisen through phage- or plasmid- mediated events.~~

~~The fact that the structure and pharmacological actions of these toxins are so similar may tend to indicate a common evolutionary origin; for~~

~~instance, a single phage/plasmid DNA sequence may have given rise to a protein molecule that, through mutation in the nucleotide sequence, has~~

~~resulted in the existence of a very closely related group of neuro toxins. It is interesting to note that diphtheria toxin is also coded~~

~~for on a bacteriophage genome (Pappenheimer, 1977). If the botulinum~~

~~neurotoxins had a similar evolutionary origin, one would expect them to~~

~~show sequence homologies in addition to the similarities in gross~~

~~structure already mentioned above. The determination of amino-acid~~

~~sequences from large proteins (about 1500 residues for BoNT) by~~

~~N-terminal analysis is not a realistic proposition; complications arise~~

~~from rapidly decreasing recoveries of polypeptide at each step,~~

~~fragmentation and increasing background signals. Proteins of large molecular weights are thus usually fragmented first and then the~~

~~peptides separated before sequencing is attempted. A prerequisite for~~

~~such studies with BoNT, is that its subunits must be prepared in~~

~~relatively large amounts in a pure and soluble form; the solubility of~~

~~the L-subunit of type A BoNT has proved problematical in its preparation~~

~~(see Chapter 2; Krysinski and Sugiyama, 1980; Sugiyama, 1980) as with~~

~~the L-subunit of type B neurotoxin, although to a lesser extent with the~~

~~latter toxin. However, having achieved the preparation of subunits in a~~

~~soluble form, it would be a monumental task to elucidate the sequence of~~

~~these large polypeptides using the above methodologies. The answer to~~

~~this problem lies in the future, probably with the determination of the~~

~~nucleotide sequence of the genes involved. Meanwhile, an indication as~~

~~to the presence of sequence homologies between proteins may be readily~~

~~determined by peptide mapping studies. Peptide maps obtained after~~

~~partial digestion of subunits from types A and B BoNT with a- chymo-~~

~~trypsin (see Chapter 3) showed significant homologies between the two~~

~~H-subunits and also both L-subunits. This was not an unexpected finding~~

~~since analogies with other toxins (e.g. cholera, tetanus, diphtheria,~~

~~abrin and ricin) suggest that one subunit is responsible for binding~~

~~activity whilst the other is effectual in the toxin’s physiological~~

~~action (Olsnes and Pi hi, 1982; van Heyningen, 1976, 1982a; Zanen et al.,~~

~~1976). If this is the case with BoNT (discussed later) then~~

~~similarities in these activities ought to be reflected in protein~~

~~sequence homologies. The demonstration of similarities in the peptide maps of H- and L-subunits of the same type of BoNT was not so~~

~~preconceived. Another peptide mapping study using reverse-phase HPLC~~

~~supported the similarities noted in peptide maps obtained by SDS-PAGE~~

~~and thus affords some significance to them. In addition, using the~~

~~compositional index S a n (Cornish-Bowden, 1983), a weak indication of~~

~~relatedness between amino acid compositions of the aforementioned pairs - 223 -~~

~~of subunits was demonstrated. Similarly, a comparison between reported~~

~~amino-acid compositions for the H- and L-subunits of type C BoNT (Syuto~~

~~and Kubo, 1981) showed "...... a strong indication, amounting almost~~

~~to certainty, that the proteins are related" (Cornish-Bowden, 1983),~~

~~4 although the former authors do not acknowledge any similarity. In view~~

~~of the phage-mediated toxigenicity of type C and D strains of Cl.~~

~~botulinum and the structural findings discussed above, is it possible~~

~~that the different subunits of botulinum neurotoxins all arose from part * of a phage genome coding for a lighter subunit (Mf *>.50000); the~~

~~heavy-subunits arising from some duplication event of the ancestral~~

~~gene The answer to this question will surely come from the elucidation 4 of the nucleotide sequences for these neurotoxins.~~

~~6.2 SEQUENTIAL STAGES IN THE ACTION OF BoNT.~~

~~4 Nearly all the information gathered so far in the interaction of~~

~~BoNT with neuronal membranes has been by indirect electrophysiological~~

~~studies on the NMJ. On the basis of such indirect measurements, it has ♦ been proposed that BoNT acts in a similar manner to other bacterial~~

~~toxins in that there are three different stages in its action; namely a~~

~~binding step followed by internalisation of the bound toxin, or at least~~

~~part of it, and an action on an intracellular target (Simpson, 1980).~~

~~The preparation of highly radioactive (800-2000 Ci/mmol) 125 I-derivatives of types A and B BoMT, that retain biological~~

~~4 activity are described here for the first time (Chapter 4). These~~

~~fully-characterised preparations have allowed considerable advances to~~

~~be made in the elucidation of the nature of BoNT acceptors in the CNS~~

~~(Chapter 5). Parallel studies at the murine NMJ using types A and B 125 I-BoNT, carried out in this laboratory (31ack, 1984), reveal many~~

similarities between the CNS and NMJ in the nature of theiracceptors for BoNT; in addition, direct evidence for an internalisation process has been demonstrated. These findings are discussed below in relation to the 3-step model proposed for the action of BoNT.

6.2.1 Toxin binding. The physiological effects of botulinum toxins in the peripheral nervous system have been known for many years (Burgen et al., 1949; Boroff et a K , 1974; Cull-Candy et al_., 1976a; Gundersen,

~~1980). However, until recently attempts to localise these toxins at the~~

NMJ were restriced to in vitro studies using fluorescein- or ferritin- labelled toxin-haemagglutinin complexes (Zacks et al_., 1962, 1968) or 125 I-labelled neurotoxins and light-microscope autoradiography

(Hirokawa and Kitamura, 1975; Dolly et a K , 1981). The specificity of silver grain depositions and biological activity of the I-BoNT (A) used in the latter study (Dolly et a1_., 1981) was confirmed when similar IOC observations were made following in vivo injection of type A I-BoNT

(Dolly et al_., 1982). The preparation of type A I-BoNT (described herein) has allowed these studies to progress to the electron-microscope level. Autoradiography of labelled synaptosome preparations from rat brain (Chapter 4) has demonstrated that the toxin is bound to the presynaptic areas of the membrane; post-synaptic densities were devoid of silver grains. A similar observation using a double-sandwich immunocytochemical technique has been reported (Hirokawa and Kitamura, 1979). 125 The autoradiographic localisation of I-BoNT on synaptosomal

~~IOC membranes corresponds with toxin binding data which showed that I-~~

BoNT (A) preferentially binds to the synaptic plasma membranes of sub- 125 fractionated synaptosomes. The localisation of type A I-BoNT at the murine NMJ by electron-microscope autoradiography demonstrated that the toxin bound saturably to all unmyelinated areas of the presynaptic membrane and that these acceptors mediate toxin internalisation

[discussed laterJ (Black et a K , 1983; Dolly et al_., 1984a); this was also observed using type B *25I-BoNT (Black, 1984). The fact that in both the CNS and the NMJ binding sites for *2^I-BoNT do not appear restricted to the membrane region opposite the post-synaptic membrane suggest that these sites may not be directly involved in transmitter release.

Synaptosomal membranes appear to contain heterogeneous populations of acceptors for types A and B A I-BoNT, as shown by equilibrium binding and kinetic methods (Chapter 4). These acceptors exhibit either high or low affinity for the toxin and are similar in nature (see below). It is interesting that the KQ determined for the high- affinity binding component is comparable to the IC^q reported for the inhibition of evoked ACh release from brain synaptosomes by botulinum toxin-haemagglutinin complex (Wonnacott, 1980a). In this context one must be reminded that preparations of cerebrocortical synaptosomes are very heterogeneous with respect to the types of synapses present; it has been estimated that only about 10% of these are cholinergic in nature (Richardson, 1981). Therefore, it is possible that the high affinity binding observed resulted from interaction of toxin with synaptic membranes from cholinergic neurons. Considering the large numbers of sialic acid containing components on all types of neuronal 125 membranes in the CNS it would not be surprising if I-BoNT exhibited a low-affinity interaction with some of these moieties (e.g. components of non-cholinergic membranes). The differences in the binding step may explain why the toxin is so much less effective at inhibiting the release of neurotransmitters such as 4-amino-butyrate compared to ACh

(Bigalke et al_., 1981). However, if the KD for BoNT is the same in the peripheral nervous system, as it is in brain, the occupancy to reactivity ratio must be very low in order for the unique potency of this toxin to be manifested. - 226 -

~~Using high concentrations of type A toxin-haemagglutinin complex~~

~~(105 mean lethal doses/ml), Holman and Spitzer (1973) showed an~~

~~inhibition of adrenergic transmission in the mouse and guinea-pig vas~~

~~deferens, although the rate of blockade was two to six times slower than~~

* that observed at the rat diaphragm; a 50% inhibition of neurotrans mission (nerve stimulated at 10Hz, 0.5msec duration) in the guinea-pig

~~and mouse vas deferens was reported after 3-5 hr incubation with toxin,~~

~~whereas at the NMJ, using similar toxin concentrations, complete * inhibition was observed (nerve stimulated at 0.2Hz, 0.1msec duration) in~~

~~%lhr (Simpson, 1980). However, Mackenzie et al_. (1982) found no~~

~~inhibition at adrenergic synapses (nerve stimulated at 0.2- 5Hz, 0.3~~

* msec duration) using concentrations of pure neurotoxins Kl O^ mouse

~~LD50/ml) known to inhibit ACh release at motor nerve terminals~~

~~(Simpson, 1982). In addition, Dolly et al_. (1984a), using type A 125 I-BoNT found no evidence for neurotoxin binding to mouse vas~~

~~deferens preparations, even at high concentrations (22nM, %2.5 x 10^~~

~~LD50/ml). The absence of a specific acceptor(s) for BoNT may explain~~

~~why neurotransmission is not generally affected at these synapses. The * reported lower potency of BoNT at adrenergic synapses (Holman and~~

~~Spitzer, 1973) or on noradrenergic transmission in the rat anococcygeus~~

~~muscle (Mackenzie et a K , 1982) may therefore result from the toxin's~~

~~♦ inefficient uptake by some non-specific route. The inability of type A 125 I-BoNT to bind to the mouse vas deferens in addition to electro-~~

~~physiological data (discussed above) suggests that non-cholinergic~~

~~synapses are not significantly affected by this toxin. The observation * that BoNT affects transmission in non-adrenergic, non-cholinergic~~

~~neurons of the guinea-pig urinary bladder (Mackenzie et a K , 1982) may~~

~~reflect co-release of ACh and a non-cholinergic transmitter(s); the~~

~~presence of large opaque vesicles in some apparently cholinergic neurons~~

~~(Hoyes et al_., 1977), structures previously associated with non- adrenergic, non-cholinergic relaxations of the guinea-pig fundic strip~~

(Paul and Cook, 1980) and taenia coli (Mackenzie et al_., 1982) are not blocked by BoNT. 125 Interaction of I-BoNT with acceptor(s) at the NMJ shows several similarities with that found in brain preparations; binding is not Ca - or temperature-dependent, although it is temperature sensitive (see Chapters 4 and 5; Dolly et al_., 1984a). The preparation of H- and L-subunits from type A BoNT has allowed their ability to compete with I-BoNT (A) for its binding sites to be examined; binding of I-BoNT (A) to synaptosomes was only inhibited by its separated

H-subunit. In addition, the H-subunit was also effective in preventing binding of type A ^^I-BoNT at the NMJ (Dolly et al_., 1984a) suggest ing that in both cases this subunit mediates the binding of type A BoNT.

~~A similar effect was found with the H-subunit of type B BoNT in inhibit- 125 ing the binding of type B I-BoNT to rat brain membranes (Kozaki,~~

~~1979); in the latter study a significant inhibition was also reported for the L-subunit, although this was probably due to contamination with~~

H-subunit or native toxin. Additional, direct evidence of H-subunit binding was demonstrated herein by the saturable interaction of a 125 I-labelled preparation of H-subunit with synaptosomal membranes.

The observed specific binding of the H-subunit from type A BoNT to synaptic membranes, together with the fact that this separated subunit is essentially non-toxic, is evidence that additional step(s) must be involved in this toxin’s action beyond the initial binding; it is likely that the L-subunit contributes to one of these additional stage(s), but evidence in support of this is presently lacking. As to the selectivity of toxin binding, with respect to the different botulinal neurotoxins, the data presented suggest that at least some of the toxins bind to independent sites. Type B BoNT was unable to compete 125 with type A I-BoNT for its specific sites although type A BoNT had a weak affinity for some acceptors for type B c I-BoNT (Chapter 5);

~~similar observations have recently been shown at the NMJ (Black, 1984).~~

~~Kozaki (1979), using brain synaptosomes, came to the same conclusions; however, in this study the binding of type B BoNT to high affinity sites~~

~~for type A toxin was not examined. In addition, Kozaki (1979)~~

~~demonstrated that types A and E BoNT appear to interact with at least~~

~~some common sites with similar affinity.~~

~~The nature of the binding component for types A and B BoNT was~~

~~investigated using synaptosome preparations as these studies were not~~

~~feasible at the NMJ. The significant inhibition of binding of both~~

~~labelled neurotoxins by preincubation of membranes with neuraminidase or wheat germ agglutinin suggest the involvement of sialic acid residues~~

in toxin-acceptor interactions. Also, the ability of neuraminidase to inhibit toxin binding with only minimal effects on neurotransmitter uptake and release (Bigalke et al_., 1981; Habermann et al_., 1981) is

~~further evidence in support of the existence of 'spare receptors'.~~

~~However, this may also indicate that the binding prevented by treatment of synaptosomes with neuraminidase is not "effective" in the toxin's~~

~~subsequent action on transmitter release; the occurrence of "effective" and "non-effective" binding (as distinguished by neuraminidase~~

~~sensitivity) of tetanus toxin has been previously proposed to account~~

~~for this toxin's action on neuroblastoma cells (Zimmermann and~~

~~Piffaretti, 1977). The possibility that gangliosides (which contain sialic acid) are involved in the neuronal binding of botulinum neuro~~

~~toxins still remains unclear. Simpson and Rapport (1971a,b) suggested that botulinum toxin may be detoxified by incubation with certain~~

~~gangliosides (especially GT^) where the number and position of sialic acid residues was of importance. More recently, Kitamura et al_. (1980)~~

reported that out of many gangliosides tested, only GTlfc> was highly effective in detoxification; this ganglioside is reported to give a 50% - 229 - detoxification of BoNT when present in a 6-fold molar excess over toxin.

However, their observation that inactive I-BoNT (prepared using excess amounts of chloramine-T in the iodination procedure) still retained its ability to complex with ganglioside tends to suggest that a 1 pc non-specific interaction occurs; in our hands, inactive I-BoNT, similarly prepared, failed to bind specifically to brain synaptosomes or at the NMJ. In addition, Wonnacott (1980) failed to find an effect of gangliosides on the inhibition of transmitter release from synaptosomes by botulinum toxin-haemagglutinin complex; neither did incorporation of gangliosides into synaptosomal membranes potentiate the action of the toxin. Thus the detoxification noted by some authors (Kitamura et al.,

1980; Simpson and Rapport, 1971a,b) and the weak competition by gangliosides described herein (see Chapter 5) may be due to some kind of non-specific inactivation of toxin with ganglioside as discussed previously (van Heyningen and Mellanby, 1973). However, it is possible that a specific ganglioside present in small amounts may be accountable for the inhibition of toxin binding observed in the competition experiments described above (Chapter 5). Moreover, Kitamura et al.

(1980) reported that their preparation of labelled toxin did not form complexes with ganglioside or bind to synaptosomes at low temperatures; this does not correspond with data from the CNS or NMJ, as discussed above (see Chapter 4; Dolly et al_., 1984a), or findings from electrophysiological investigations (Simpson, 1980), which suggest that the functional binding of BoNT (i.e. that which results in toxicity) is not temperature-dependent. 125 The inhibition of types A and B I-BoNT binding to synap tosomes observed after their preincubation with some phospholipases Ag indicates a requirement for specific membrane phospholipids in toxin binding; whether this inhibition is due to lipolysis of the acceptors per se or to a general perturbation of the membrane structure by the enzymes is presently unknown.

The only significant differences noted in the nature of acceptors for types A and B BoNT were in their sensitivities to trypsinisation or heat-denaturation. Either treatment of synaptosomal membranes completely abolished their ability to specifically bind type A 125 I-BoNT whereas similar treatments only partially prevented type 3 125 I-BoMT binding. This suggests the involvement of proteinaceous components in the majority of binding of both toxins. However, in the case of type B BoNT some of its specific sites are either lacking in protein or these are not susceptible to proteolytic cleavage or heat denaturation; at least the heat-resistant sites appear to bind toxin with high affinity. Bearing in mind the negative effect of high energy radiation on the high- or low-affinity synaptosomal acceptors for types 125 A and B I-BoNT, it appears that whatever the nature of these membraneous acceptors is they are of relatively small molecular size.

The physiological function(s) of these neuronal acceptors for BoNT is, to date, unknown; however, it is highly unlikely that.they have evolved specifically to accommodate these toxins. Externally located glycosylated molecules may be important in cell-cell recognition and interaction, neuronal differentiation or perhaps more indirectly in the mechanism(s) of neurotransmission. The negative charge of sialic acid residues (abundant in neuronal membranes) may play a fundamental role in ensuring a high local concentration of cations in close proximity to the extracellular plane of the membrane; this may facilitate rapid and efficient depolarisation of the neuron on stimulation.

6.2.2 Internalisation. The first indication that toxin binding is not directly responsible for the neurotoxic action of botulinum toxin came from studies by Burgen £t al_. (1949). These workers showed that removal of excess toxin (during the lag phase of the toxin's action) from incubations with mouse nerve-muscle hemidiaphragms did not prevent subsequent paralysis by the toxin. Since then, Simpson (1980), using indirect electrophysiological methods, has provided evidence for the involvement of three steps in the action of botulinum toxin; the additional step being receptor-mediated internalisation. It was reported that toxin could be maintained at an anti-toxin sensitive site in the absence of physiological concentrations of Ca2+ ions and that internalisation was accelerated by nerve-stimulation. Recently, this process of toxin internalisation has been demonstrated directly using types A (Dolly et a K , 1984a) and B (Black, 1984) 125I-BoNT and electron-microscope autoradiography. Here it was shown that internal isation was an energy-dependent process being prevented by incubation at

4°C or when binding was performed in the presence of metabolic inhibitors (e.g. sodium azide or dinitrophenol). Under the latter IOC conditions it was shown clearly that type A I-BoNT bound to all unmyelinated areas of the presynaptic nerve-terminal; it also allowed the estimation of the number of peripheral binding sites as 150- 500 per o pm of plasma membrane (Dolly et al_., 1984a). These findings establish that the binding step is essential for internalisation.

~~The exact method by which the toxin is internalised is unclear.~~

An obvious choice would be endocytosis since this process occurs in all cell types (Silverstein et a K , 1977) and it is known that increased nerve stimulation results in increased rates of endocytosis (Heuser and

Reese, 1973). Is it just a coincidence that increased nerve stimulation also results in the more rapid development of the pharmacological actions of BoNT ?. Another mode of toxin entry into cells, other than endocytosis, has been hypothesised for at least some bacterial toxins

(Gill, 1978). It has been proposed that part of the toxin may be inserted into the membrane structure where it may form a protein carrier or channel allowing subsequent direct penetration of the membrane by the remainder of the toxin molecule. Diphtheria toxin (or its A-fragment) is reported to be able to penetrate directly through the cell membrane at low pH (Sandvig and Olsnes, 1980). However, the fact that both sub units of this toxin may penetrate the membrane (Wisnieski and Zalman,

1983) and that at neutral pH when endocytosis is prevented with meta bolic inhibitors, cells are protected against the acion of diphtheria toxin (Sandvig and Olsnes, 1982a), suggest that endocytosis is a step in the normal internalisation process of this toxin. Employing photolabelling techniques, Ishida elt al_. (1983) have shown that both ricin

A- and B-chains may penetrate membrane bilayers and that reduction of the inter-subunit disulphide bond greatly enhances this penetration. A similar observation with reduced diphtheria toxin has also been reported

(Wisnieski and Zalman, 1983). The similarities between the penetration of membranes by ricin and diphtheria toxin do not appear to be compatible with the situation found with botulinum neurotoxins, where it is well documented that reduction of the inter-subunit disulphide bridge of BoNT causes detoxification (Sugiyama et al_., 1973). However, if this inactivation is due to the inability of the reduced toxin to bind its acceptors, it is possible that extracellular reduction may possibly occur after the toxin has bound.

In contrast to the energy-dependent internalisation of type A 125 I-BoNT at the NMJ, negligible internalisation of bound toxin was observed in rat brain synaptosome preparations (Chapter 4, Fig. 4.11). 125 The apparent inability of synaptosomes to internalise type A I-

~~BoNT may account for the much decreased potency of BoNT in the central nervous system in vitro (Habermann, 1981) as compared to the periphery.~~

The effects of BoNT on transmitter release from synaptosomes may then be attributed to toxin molecules internalised by some other inefficient or relatively non-specific route. However, localisation studies involving the use of brain slices or in vivo application of I- BoNT centrally must be performed to ascertain whether this observed lack of Internal isation Is artlfactual of the synaptosome preparation used. In addition, one must note that various cell types exhibit differential rates of internalisation (Heuser and Reese, 1973; Zimmerman, 1979) and thus a slow rate of internalisation may not be readily apparent.

6.2.3 Toxin-induced inhibition of neurotransmitter release. The presynaptic pharmacological actions of botulinum toxins on the motor nerve terminal have been well characterised (see General Introduction; reviewed by Simpson, 1981a) and even some post-synaptic effects of BoNT have been reported (Sellin, 1981); however, these latter findings are probably an indirect result of the denervation-like effects of botulinum toxin on the NMJ. This assumption is supported by the lack of evidence 1 pc for any post-synaptic binding of I-BoNT (A or B) at the NMJ (Black,

~~1984; Dolly et al_., 1984a).~~

~~In contrast to the unique in vivo potency of BoNT in the inhibition of ACh release at the NMJ, toxin concentrations as high as~~

~~10^-10® mouse LOgg/ml are required to block release of this transmitter in vitro; lower concentrations (3 x 10 mouse LD^/ml =~~

10-11M) are reported to be effective although they exhibit a much pro longed time course of action (Simpson, 1980). This does not necessarily imply that acceptors for BoNT at the NMJ are not of high affinity, as even higher concentrations KlpM) of a-BuTx (KQ %10” **M) are required to overcome diffusion barriers in this tissue under similar incubation conditions (Dolly et al_., 1977).

A physiological action of botulinum toxins in the CNS is less clearly defined than it is at the NMJ. In contrast to early reports that botulinum toxin-haemagglutinin complexes exhibited a central action

(Polley £t al_., 1965; Wiegand and Wellhoner, 1977), purified BoMT has not yet been shown to be toxic in the brain (in vivo). Intraventricular injection of relatively high amounts of type A BoNT (up to 5000 mouse

LD^q ) into rats showed no apparent toxic effects centrally (Vlilliams, et al., 1983). However, botulinum toxin complexes have been shown to partially inhibit evoked release of ACh from rat brain synaptosomes

(Wonnacott and Marchbanks, 1976; Wonnacott, 1980a); also, purified BoNT has been shown to inhibit resting and evoked transmitter release from such preparations (Bigalke et al_., 1981; Dolly et al_., 1981). The function of the specific synaptosomal acceptors for types A and B i pc I-BoNT described herein (Chapters 4 and 5), in relation to the toxins' action(s) on neurotransmitter release is not absolutely clear.

The many similarities in toxin binding characteristics between the CNS and the NMJ suggest that these similar sites may be identical or at least very similar in nature. At present there is no evidence for high- and low-affinity acceptors for BoNT at the NMJ, although the method ologies available at present may not be able to distinguish these sites.

The reasons for the lack of toxicity in the brain in vivo may be owed to the apparent inability of central synapses to effectively internalise bound toxin, or the absence (or modification) of any one component or process necessary for the manifestation of its neurotoxicity once internalised. If internalisation of BoNT in central neurones occurs via a general mechanism (e.g. adsorptive endocytosis), then high-affinity binding of the toxin to the membranes may greatly facilitate its entry by such a route. Therefore, if the observed high-affinity binding of

BoNT to synaptosomes represents interactions with membranes of cholinergic neurons, this may account for the toxin’s more potent inhibition of ACh release compared with other transmitters (Bigalke et al_., 1981).

Many physiologically active compounds such as guanidinuim hydro chloride (Cherington, 1980), 4-ami nopyridine (Ball et al_. , 1980; Lundh et al., 1977) and various lysosomotropic agents (Simpson, 1982) have been tested in search of an antagonist for botulinum neurotoxin. Of those reported, ami noquinolines appear to be the most effective

(Simpson, 1982); chloroquine delays the onset of paralysis by botulinum toxin but it was not demonstrated whether this effect was at the binding, internalisation or subsequent step(s) of the toxin's action.

~~It has now been shown that chloroquine does not affect the binding of 125 type A I-BoNT to rat brain synaptosomes (see Chapter 5) or to the~~

NMJ (Dolly et al_., 1984a). This latter study, at motor nerve terminals, also demonstrated that chloroquine is unable to prevent toxin internal isation. Therefore, it is likely that chloroquine, being a potent lysosomotropic agent (de Duve, 1983), acts at an internal site, possibly affecting the lysosomal or other compartmentalised (e.g. endosomal) processing of the toxin molecule, as proposed for diphtheria toxin

~~(Leppl a et a K , 1980).~~

~~6.3 SIMILARITIES WITH TETANUS TOXIN.~~

~~Toxigenic strains of Cl. tetani produce apparently only one type of tetanus toxin as compared to the variety of neurotoxins produced by~~

Cl. botulinum species. This protein may be synthesised as a non-toxic variant, thought to differ in only a few amino-acid residues from the active form (cf. Wellhoner, 1982); such a variant has not been demonstrated in cultures of Cl. botulinum. However, non-toxigenic strains of both Clostridial species have been reported (Hara et al.,

~~1977; Sugiyama, 1980).~~

A comparison of the molecular structures of botulinum (generally speaking) and tetanus neurotoxins reveal striking similarities. They are synthesised by species of Clostridia and may possibly be encoded on phage or plasmid genomes (Eklund and Poysky, 1981; Laird et al_., 1980); both types of protein have an Mr %150000, consisting of two subunits (Mr 'v.lOOOOO and 50000) linked by one or more disulphide bonds. The neurotoxins are synthesised as a single polypeptide, subsequently being nicked by endogenous proteases to form dichain molecules (reviewed by

Sugiyama, 1980; WellhiJner, 1982). Mild treatment of the toxins with papain (tetanus toxin) or chymotrypsin (BoNT) results in the formation of two polypeptides; an ^-fragment (Mr %50000) which is half of the H-subunit and an Hg-L fragment (Mr ^100000) containing the other half of the H-subunit linked by a disulphide bond(s) to the L-subunit

(cf. Sugiyama, 1980). The two subunits of tetanus toxin are shown to be antigenically distinct (Matsuda and Yoneda, 1975) as described above for various botulinum neurotoxins. Despite these immunological differences, similarities between the structures of H- and L-subunits from tetanus toxin have been reported (Taylor et a K , 1983); there is also some indication of similarities between the subunits of types A and B botulinum neurotoxins (see Chapter 3).

The interaction of both neurotoxins at the NMJ and in the CNS has been studied in some depth (Dreyer and Schmitt, 1981, 1983; Dreyer et al., 1983; Habermann, 1981; Simpson, 1981a; Wellhoner, 1982). As described in Chapters 4 and 5 for type A and B botulinum neurotoxins, tetanus toxin may also bind to brain particulate preparations with relatively high affinity (Rogers and Snyder, 1981) and binding of this toxin is reported to be mediated by its larger subunit (van Heyningen,

1976). From early studies using I-labelled botulinum toxin of low specific radioactivity, Habermann (cf. 1981) reported that tetanus toxin and BoNT (A) bind to different sites. However, it has now been demonstrated that tetanus toxin has a low-affinity for the majority of acceptors for type A BoNT in the CNS (Chapter 5) and at the NMJ (Dolly et al., 1984a). Moreover, tetanus toxin was found to significantly inhibit the binding of both types A and B 125I-BoMT to their high affinity acceptors) on synaptosomal membranes (Chapter 5); the differences In nature observed between some of the aforementioned sites for types A and B BoNT (1.e. heat and trypsin sensitivities; see previous chapter) and the reported resistance of acceptors for tetanus toxin to protease treatment (Rogers and Snyder, 1981) suggest that these toxins bind preferentially to different neuronal components.

~~However, the sensitivity of acceptors for tetanus, and types of A and B botullnum toxins to neuraminidase (see Chapter 5; Habermann and Heller~~

1975; Rogers and Snyder, 1981) suggests the overall importance of sialic acid residues in binding. Tetanus toxin has been known for many years to bind to specific gang!iosides (van Heyningen and Mellanby,

~~1973); a high-affinity interaction with gangliosides GD^a , G D ^ and~~

~~GTlb has been reported (Helting et a K , 1977; Rogers and Snyder,~~

1981; van Heyningen, 1976). Whether such binding of tetanus toxin to gangliosides in vivo results in the subsequent pharmacological actions of this toxin remains to be established (see below). As mentioned above, the binding of tetanus toxin to its acceptors in brain tissue is neuraminidase sensitive; nevertheless, pretreatment of synaptosomes with this enzyme is ineffective in preventing the toxin's inhibitory action on K+-evoked ACh release (Bigalke et al_., 1981). This is entirely analogous to the data reported for BoNT (discussed above).

Possible explanations for these data are that two populations of receptors are present or that spare receptors are involved in the toxin binding observed. There is at least some evidence to support the idea that different acceptor populations may exist. Tetanus toxin binds to mouse neuroblastoma cells in growth culture in a neuraminidase- sensitive manner; in differentiating cultures, neuraminidase treatment of cells has no effect on toxin binding or its physiological effects

(Zimmerman and Piffaretti, 1977). These authors suggest that toxin fixation may occur other than by binding to gangliosides and that, at least in the case of differentiating cultures, interaction with gangliosides may not be related to any biological effect. In cell cultures of

~~cerebral neurons Yavin et a K (1983) reported that gangliosides are all~~

~~important in binding of tetanus toxin and its temperature-dependent~~

~~internalisation event. Owing to the ability of chloroquine and other~~

~~lysosomotropic agents to significantly delay onset of paralysis caused~~

~~by BoNT, it is likely that the toxin's internalisation at the NMJ occurs~~

~~through an endocytotic mechanism (discussed above). However,~~

~~chloroquine is not effective in altering the susceptibility of neurons~~

~~to tetanus toxin, indicating that endocytosis is not involved in the~~

~~normal route taken by this protein in its course of action (Simpson,~~

~~1982). In view of the varied data reported so far on the binding-~~

~~function relationships of botulism and tetanus toxins, further~~

~~investigations are required to elucidate the specific binding which is~~

~~directly responsible for the pharmacological actions of these toxins.~~

~~Superficially botulinum and tetanus toxins apear to have~~

~~completely different modes of action suggested by their characteristic~~

~~syndromes of flaccid and spastic paralysis, respectively. On detailed~~

~~investigation, both neurotoxins were found to inhibit uptake and release~~

~~of neurotransmitters at the NMJ (Habermann, 1981) and in the CNS~~

~~(Bigalke et al_., 1978, 1981; Habermann, 1981). However, tetanus toxin was highly potent in blocking glycine-mediated inhibition in~~

~~preparations from brain and spinal cord; whereas type A botulinum toxin~~

~~had negligible effects (Bigalke et a K , 1981). To date, there is no~~

~~evidence that either toxin affects peripheral noradrenergic synapses~~

~~(Dolly et a K , 1984a; Habermann, 1981; Mackenzie et al_., 1982). The~~

~~differences that occur in the actions of these toxins have, until~~

~~recently, been put down to quantitative rather than qualitative effects;~~

hence, tetanus toxin was reported to exert almost identical pharmaco logical actions to BoNT at the NMJ except that the latter toxin is about one thousand times more potent (Habermann, 1981). There are numerous reasons why these toxins exhibit different potencies at peripheral and central synapses; these include possible differences in their binding affinities, their mode and efficiency of internalisation and intra cellular processing (e.g. within lysosomes or endosomes). However, electrophysiological data, obtained from studying the effect of

4-ami nopyridine and nerve stimulation on quantal transmitter release after poisoning with BoNT (A) or tetanus toxin, suggests that these toxins act at different sites in the stimulation-secretion process at the NMJ (Dreyer and Schmitt, 1981). More recently, Dreyer and Schmitt

(1983) have reported further differences in the electrophysiological effects of these two toxins. Namely, these include the ability of single nerve stimuli to elicit epps in botulinised muscles, but not in tetanus toxin-treated preparations; an effect of tetanus toxin (but not

BoNT) that resulted in an asynchronous quantal release of ACh and the ability of tetanus toxin to preferentially block the release of large amplitude mepps without affecting small amplitude ones; BoNT inhibited both large and small amplitude mepps. These differences besides, the similarities between botulinum and tetanus toxins in their basic structure, neuronal binding, internalisation and paralytic characteristics remain very striking. Considering the asynchronous release of ACh reported in type B botulinum toxin-treated nerve-muscle preparations (Thesleff, 1984), perhaps in the future, this toxin (and maybe other types of BoNT) will be classified as another form of tetanus toxin, or vice versa.

~~6.4 FUTURE PERSPECTIVES~~

The mouse LD^g test used to determine the toxicity of botulinum toxin samples is far from ideal; apart from the large numbers of animals that have to be sacrificed, it is not a particularly accurate means of assay. The purification of BoNT from its complexes with

~~haemagglutinin and other non-toxic proteins has allowed antibodies to be~~

~~raised against the neurotoxin; this in turn has permitted the~~

~~development of a more sensitive and reliable enzyme-linked immunosorbe-~~

~~sorbent-assay (ELISA) for the neurotoxin (A.C. Ashton - unpublished~~

~~data). In addition, the radiolabelling of types A and B BoMT to high~~

~~specific activities has enabled the development of a very sensitive~~

~~radioimmunoassay, as an alternative to ELISA (Ashton et al_., 1984). If~~

~~the different types of BoNT contain conserved amino-acid sequences it may be possible to produce a single antitoxin for all types (or at least~~

~~several of them); at present, each antitoxin has to be prepared~~

~~individually. Toxoids used in the preparation of antibodies are made by~~

~~prolonged incubation of the toxin with formaldehyde to effect complete,~~

~~irreversible inactivation. It has been shown that irreversible~~

~~alkylation of thiols in the reduced BoNT molecule results in loss of~~

~~biological activity, as also does the separation of the H- and L-sub~~

~~units. Possibly in the future, various chemical modifications of the~~

~~intact toxin or its individual polypeptides (or a fragment of them) may~~

~~allow the production of more efficient and specific toxoids. This may~~

~~allow anti-toxins active at various stages in the course of this toxin's~~

~~action to be obtained; to date this has not been achieved.~~

~~It will be of great interest to compare the amino-acid sequences~~

~~of the polypeptides from the different types of BoNT; such a comparison~~

~~may show highly conserved regions of sequence that may give an~~

~~indication to the areas of the protein directly involved in the~~

~~different stages of the toxin’s action. This goal will probably only be~~

~~reached by sequencing the genes for these different toxin types. Before~~

~~this is possible a limited amount of protein sequence data must be~~

~~obtained from toxin subunits to enable the synthesis of specific nucleo~~

tide probes. The area of research on BoNT where significant advances have yet to be made, as with most other toxins, is that of relating the membrane binding activity of the toxin to its subsequent pharmacological action.

At present it is not known whether either or both of the subunits penetrate the membrane and whether such penetration is direct or via an endocytotic mechanism. There is evidence to support the latter method of entry at the NMJ, although it is quite possible that both processes occur within the same cell. A recent approach to determine whether either or both subunits of a protein/toxin may penetrate a membrane utilises photoreactive lipid probes to label proteins within the lipid core of the membrane. Photolabelling has allowed significant advances to be made in the knowledge of which subunits of ricin and diphtheria toxins are capable of penetrating biological membranes (Ishida et al.,

~~1983; Wisnieski and Zalman, 1983). This technique may be applied to~~

~~BoNT to elucidate its mode of insertion into and/or through membranes.~~

The only membrane model available at present is that of rat cerebrocortical synaptosomes; if penetration through these membranes is non specific compared to that at the NMJ then its physiological significance would be held in doubt. On the other hand, if internalisation of toxin into synaptosomes is a specific process, at least from the high-affinity binding sites observed, then such invesigations might shed some light on what may be happening at the NMJ. In the course of these studies reported herein, various other systems have been investigated for their

~~i pc ability to interact specifically with I-BoNT. These included preparations from the cholinergically-innervated electric organs of~~

Torpedo melanogaska (sting ray) and Electrophorus electricus (electric eel), myenteric plexus synaptosomes and cell cultures (PC12 primary cell cultures and neuroblastoma-glioma cultures). However, so far, none of these models for cholinergic neurotransmission, under the conditions used, have proved successful in the study of either toxin-acceptor interactions or the toxin's subsequent blockade of transmitter release.

Another exciting area of research developing rapidly nowadays is that of the production of chimeric toxins. These artifical hybrid proteins may contain a receptor-binding specificity of one toxin and the effector function of another, thus allowing them to exert their effect through alternative receptors and hence different specific cell types.

BoNT hybrids should provide more information regarding specific cellular binding sites and their role in toxin internalisation; also the specific requirements for interactions between subunits may be defined.

Such chimeric proteins have already been made from the A- subunit of diphtheria toxin or ricin together with a variety of lectins, other heterologous toxin subunits and antibodies (Gilliland et a K , 1981;

~~Jansen et al_., 1981; Neville et al_., 1981; Oeltmann and Forbes, 1981).~~

The advent of monoclonal antibodies has greatly improved the specificity of immuno-toxins as targeting molecules and now they hold enormous potential for therapeutic use. The H-subunit from BoNT may possibly be used as a specific probe for cholinergic synapses; a marker for presynaptic cholinergic membranes has not yet been reported. If the L- subunit of BoNT is responsible for the toxin's pharmacological actions then a hybrid of more clinical therapeutic use may possibly be obtained by linking the H-subunit of BoNT to an anti-L-subunit antibody. This molecule may then be bound and internalised in the same manner as the intact neurotoxin and delivered to the physiologically-active site of the L-subunit. Ideally, the antibody may then bind to the L-subunit, causing its inactivation and thus recovery from botulinisation.

Although there is an enormous amount of supposition in such a scheme, there is no doubt that in the future, chimeric molecules will be very important in directing specific compounds to discrete intracellular compartments (e.g. golgi apparatus, GERL, nucleus) in specific cell types (e.g. carcinoma cells). . - 243 -

~~APPENDIX~~

~~Materi al Source~~

~~DEAE-Sephadex A50 Pharmacia ii * DEAE-Sephacel CH-Sepharose 4B ii Sephadex G-25 (superfine) ii QAE-Sephadex H Agarose it Gradient pore (PAA, 4/30) polyacrylamide gels n Pharmalytes ii~~

~~Protein molecular weight markers Biorad, Pharmacia~~

~~Acrylamide, bis-acrylamide BDH~~

~~Hydrochloric acid ("Aristar" grade) BDH~~

~~% TEMED BDH~~

~~Coomassie blue R-250 and G-250 Sigma~~

~~Chiorami ne-T Hopkin and Williams Limited, Chadwell, Essex. ♦ DTT Biorad, Sigma~~

~~SDS Biorad, Sigma~~

~~Urea (ultrapure grade) Sigma * Staph, aureus Y8 protease Miles, Sigma~~

~~Guanidine hydrochloride (ultrapure grade) Bethesda Research Laboratories, Cambridge.~~

~~% Radioisotopes Amersham Internatl., Amersham.~~

~~Cultures from types A (NCTC 2916) Provided by Vaccine and B (Okra) Cl. botulinum Research and Production Unit, P.H.L.S., Porton * Down, Wiltshire.~~

~~Horse anti-(type A or B toxin-haemagglutinin n complex) antibody.~~

~~All other chemicals and reagents were analytical Sigma, BDH, Fisons, grade. Calbi ochem-Behri ng Corporation. REFERENCES~~

~~Abe,. T., Alenia, S. and Miledi, R. (1977) Eur. J. Biochem. 80, 1-12.~~

~~Abe, T., Limbrick, A.R. and Miledi, R. (1976) Proc. R. Soc. (Lond.) B. 194, 545-553.~~

~~Abrams, A., Kegeles, G. and Hottle, G.A. (1946) J. B1ol. Chem. 164, 63- 79.~~

Albuquerque, E.X. and Daly, J.W. (1977) in The specificity and action of Animal, Bacterial and Plant Toxins (Receptors and recognition) (Cuatrecasas, P., ed.) Ser. B, Vol. F , Chap. 4, pp. 129-173. Chapman and Hall, London.

~~Arnon, S.S. (1980) Ann. Rev. Med. 31, 541-566.~~

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